Integrated organ-on-chip systems and applications of the same

ABSTRACT

In one aspect of the invention, an integrated bio-object microfluidics chip includes a fluidic network having a plurality of inlets for providing a plurality of fluids, a plurality of outlets, a bio-object chamber for accommodating at least one bio-object, a plurality of fluidic switches, and one or more pumps, coupled to each other such that at least one fluidic switch operably and selectively receives one fluid from a corresponding inlet and routes the received fluid, through the one or more pumps, to the bio-object chamber so as to perfuse the at least one bio-object therein, and one of the downstream fluidic switches selectively delivers an effluent of the at least one bio-object responsive to the perfusion to a predetermined outlet destination, or to the at least one fluidic switch for recirculation.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to and the benefit of, pursuant to 35U.S.C. §119(e), U.S. provisional patent application Ser. No. 61/569,145,filed on Dec. 9, 2011, entitled “PERFUSION CONTROLLER, MICROCLINICALANALYZER AND APPLICATIONS OF THE SAME”, by John P. Wikswo et al., U.S.provisional patent application Ser. No. 61/697,204, filed on Sep. 5,2012, entitled “INTELLIGENT CHIP CARRIER AND CHIP CARRIER WITHMICROCHEMICAL ANALYZER AND APPLICATIONS OF THE SAME”, by John P. Wikswoet al., and U.S. provisional patent application Ser. No. 61/717,441,filed on Oct. 23, 2012, entitled “INTEGRATED ORGAN MICROFLUIDICS (IOM)CHIP AND APPLICATIONS OF SAME”, by John P. Wikswo et al. Each of theabove-identified applications is incorporated herein in its entirety byreference.

Some references, which may include patents, patent applications, andvarious publications, are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the inventiondescribed herein. All references cited and discussed in thisspecification are incorporated herein by reference in their entiretiesand to the same extent as if each reference was individuallyincorporated by reference. In terms of notation, hereinafter, “[n]”represents the nth reference cited in the reference list. For example,[3] represents the 3rd reference cited in the reference list, namely,Lima, E, Snider, R, Reiserer, R, Cliffel, D, Wikswo, J P. MultichamberMultipotentiostat System for Cellular Microphysiometry, Rev. Sci.Instrum., In preparation, 2011.

STATEMENT AS TO RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under grant numbersNIH/NCI R21 CA126728-01A1, NIH/NIDA RC2DA028981-02, and NIH1UH2-TR000491-01, awarded by the National Institutes of Health, DTRAgrant HDTRA1-09-1-00-13, awarded by the Defense Threat Reduction Agency,and DARPA contract DARPA-11-73-MPSys-FP-11, awarded by the DefenseAdvanced Research Projects Agency, DOD/BCRP W81XWH-10-1-0157, awarded byDepartment of Defense Breast Cancer Research Program. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to a microfluidic system, andmore particularly to perfusion controllers, microclinical analyzers,integrated bio-object microfluidics chips and systems utilizing theperfusion controllers and the microclinical analyzers and applicationsof the same.

BACKGROUND INFORMATION

Organs-on-chips are a promising means to test drug efficacy andinteractions without the need for animal testing. However, there hasbeen little thought into how multiple organ systems should be integratedto study multi-organ physiology. This invention addresses key issues inthe measurement and control of multiple organ-on-chip systems.

The measurement systems, microfabricated devices, and analytical andmodeling techniques developed over the past decade to instrument andcontrol cancer, immune, yeast, and cardiac cells provide a uniqueopportunity to address some of the most fundamental issues in organinteractions and drug responses. This problem clearly requires acoordinated, interdisciplinary, high-technology approach, such asunderstanding the interaction between lung function and organoxygenation, neuroimmune interactions, response to neural injury,cardiac arrhythmias, and development of new multimodal therapies. Todate, there have been no demonstrations of methods for controlling andanalyzing multiple organs-on-chips, particularly in a manner that allowsa single design of controller and analyzer to be dynamically configuredfor a particular application or analysis.

Therefore, a heretofore unaddressed need exists in the art to addressthe aforementioned deficiencies and inadequacies.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a perfusion controller usablefor maintenance and analysis associating with a plurality ofbio-objects, where each bio-object includes an organ or a group ofcells. In one embodiment, the perfusion controller has a plurality ofinlets for providing a plurality of fluids, a plurality of outlets, anda fluidic network coupled between the plurality of inlets and theplurality of outlets and being in fluid communication with the pluralityof bio-objects. The fluidic network comprises a plurality of fluidicswitches and one or more on-chip pumps adapted for selectively andindividually perfusing at least one of the plurality of bio-objects withat least one of the plurality of fluids at a predetermined perfusionflow rate and delivering an effluent of the at least one bio-objectresponsive to the perfusion to a predetermined one of the plurality ofoutlets, where the plurality of outlets is coupled to at least one of ananalyzer, a waste port, one of the plurality of bio-objects, and thefluidic switch network.

In one embodiment, the desired fluids contain a dye, a drug, a medium orthe like.

In one embodiment, the perfusion controller further includes a perfusionreservoir having a plurality of containers for containing the pluralityof fluids, respectively, where the plurality of containers is coupled tothe plurality of inlets for respectively providing the plurality offluids.

In one embodiment, the perfusion controller may also have amicrocontroller for individually controlling a flow rate of each fluidicpath.

In one embodiment, the perfusion controller further includes one or moresensors at least coupled to the at least one bio-object for measuring apressure drop across the at least one bio-object perfused with the atleast one fluid, so as to regulate the flow rate of the at least onefluid through the at least one bio-object at the predetermined perfusionrate.

In one embodiment, the fluidic network further comprises a plurality offluidic paths in fluid communication with the plurality of fluidicswitches and the one or more on-chip pumps, where each bio-object isdisposed in a corresponding fluidic path.

In one embodiment, each fluidic switch comprises a valve having at leastone pole and a plurality of throws, where the at least one pole isselectively operable in fluid communication with one of the plurality ofthrows.

In one embodiment, the plurality of throws of the first fluidic switchof the fluidic switch network is respectively coupled to the pluralityof inlets for respectively receiving the plurality of fluids therefrom.The plurality of throws of the last fluidic switch of the fluidic switchnetwork is respectively coupled to the plurality of outlets forselectively delivering the effluent of the at least one bio-objectresponsive to the perfusion to the predetermined outlet.

In one embodiment, the fluidic network comprises first, second, andthird fluidic switches and an on-chip pump, where the first fluidicswitch comprises a one-pole and four-throw valve, the second fluidicswitch comprises a two-pole and three-throw valve, and the third fluidicswitch comprises a one-pole and four-throw valve. In one embodiment, theplurality of bio-objects includes organ N−1, organ N, and organ N+1,where the organ N−1 is coupled to the second fluidic switch, the organ Nis coupled between the second fluidic switch and the on-chip pump thatin turn is coupled to the third fluidic switches, and the organ N+1 iscoupled to the second and third fluidic switches.

In one embodiment, the organs can be connected in parallel, where thefluidic pumps and switches provide the requisite capabilities forcontrolling the organs as stated above, but allow the organs to beconnected in parallel.

In one embodiment, the selected organs can be connected in parallelwhile other organs can be connected in series, with the seriescombination being in parallel to other organs, e.g., a gastrointestinalorgan being upstream from a liver organ, with the pair being in parallelwith, for example, the kidney and other organs. In another example, theheart can be in series with the lung, or the lung can be fluidicallybetween the right heart and the left heart. In each case, the fluidicpumps and switches provide the requisite capabilities for controllingthe organs as stated above, but allow the organs to be connected ineither series or parallel.

In one embodiment, each fluidic switch comprises a rotary planar valve(RPV) and each on-chip pump comprises a rotary planar peristalticmicropump (RPPM). Each of the RPV and the RPPM comprises anactuator/ball bearing having a circular ball-bearing cage defining aplurality of spaced-apart openings thereon, and a plurality of ballsaccommodated in the plurality of spaced-apart openings. In oneembodiment, the number of the plurality of balls is same as that ofplurality of spaced-apart openings of the circular ball-bearing cage,such that each opening of the circular ball-bearing cage accommodates arespective ball. In another embodiment, the number of the plurality ofballs is less than that of plurality of spaced-apart openings of thecircular ball-bearing cage, such that at least one opening accommodatesno ball. In one embodiment, the plurality of spaced-apart openings isspaced-equally defined on the circular ball-bearing cage, where each twoadjacent openings through the center of the circular ball-bearing cagedefine an angle θ=2π/K, K being the number of the plurality of equallyspaced-apart openings. In the embodiments, the ball bearing comprises acage and multiple balls trapped in the cage.

In another embodiment, the ball bearing has a cage and a single balltrapped in the cage.

In one embodiment, the actuator comprises a wheel defining a pluralityof spaced-apart sockets thereon in a circle, and a plurality of rollersaccommodated in the plurality of spaced-apart sockets such that arotation of the wheel causes the plurality of rollers to rotate alongthe circle.

In another embodiment, the actuator comprises a cam, and a plurality ofcam-followers engaged with the cam such that a rotation of the camcauses the plurality of cam-followers to rotate along a circular path.

In one embodiment, the RPV further comprises a plurality of selectivelycontrollable channels positioned under the actuator in relation to theplurality of equally spaced-apart openings such that at least oneselectively controllable channel is positioned under the at least oneno-ball opening or no-ball location of the circular ball-bearing cagethat does not contain a ball so that a fluid flow is allowed through theat least one selectively controllable channel, while the otherselectively controllable channels are respectively positioned under thecage openings having the ball so that no fluid flows are allowed throughthe other selectively controllable channels, where when rotating theactuator by a desired angle of (k×θ), k being 1, 2, . . . K, the atleast one no-ball-bearing cage opening is selectively placed over adesired one of the selectively controllable channels. In anotherembodiment, the cage does not have an opening at the location where noball is required.

In one embodiment, it would be possible to achieve these same functionswith ball bearings were located at other-than-equal angular spacings.

In one embodiment, the plurality of selectively controllable channelscomprises three selectively controllable channels connected in a T-likejunction, and the actuator is configured such that when rotating by adesired angle of (k×θ), two of the three channels are in fluidcommunication with each other, while the other channel is closed. Inanother embodiment, the plurality of selectively controllable channelscomprises four selectively controllable channels connected to corners ofa square fluidic path, and the actuator is configured such that whenrotating by the desired angle of (k×θ), the first and second channelsare in fluid communication with each other through the top portion ofthe square fluidic path, and the third and fourth channels are in fluidcommunication with each other through the bottom portion of the squarefluidic path, or the first and fourth channels are in fluidcommunication with each other through the left portion of the squarefluidic path, and the second and third channels are in fluidcommunication with each other through the right portion of the squarefluidic path.

In one embodiment, the RPV further comprises at least one always-openchannel positioned under the actuator in offset from the plurality ofequally spaced-apart openings, such that the at least one offset channelis in fluid communication with the at least one selectively controllablechannel under the at least one no-ball bearing-cage opening, and theother selectively controllable channels under the openings having theball bearings are closed.

In one embodiment, the ball bearings, i.e., balls and separate cages, inall these RPPM and RPV embodiments below are replaced by one or moreintegral ball-bearing cam followers arranged in a circle around themotor axis such that rotation of the motor will cause the ball-bearingcam followers to roll in a circle above a microfluidic channel, andthereby pump fluid peristaltically along that channel in an RPPM orocclude channels in an RPV.

In one embodiment, the caged balls in RPPMs that provide peristalticpumping in all these embodiments are replaced by one or more rotary,cylindrical ball bearings whose axis is at a 45 degree or other anglewith respect to axis by which the rotary ball bearing is driven in acircle, which in turn is perpendicular to the surface of themicrofluidic device that contains the channels thorough with fluid isflowing.

In one embodiment, the caged balls that provide valve actuation in theRPVs in all these embodiments are replaced by the combination of arotary actuator and a circular array of radial levers and springs suchthat the springs cause the channels beneath the levers to be compressedunless the rotary actuator is depressing the lever, thereby creating aset of normally closed valves that are opened by the rotary actuator. Inthis type of embodiment, the rotary actuator can either be one or moreball-bearing cam followers, or one of more angled cylindrical rotaryball bearings.

In one embodiment, each of the at least one always-open channel and theplurality of selectively controllable channels has an end connected toan arc fluidic path or a circular fluidic path. In another embodiment,the at least one always-open channel has first and second always-openchannels positioned under the actuator in offset from the plurality ofequally spaced-apart openings, where the plurality of selectivelycontrollable channels comprise a first plurality of selectivelycontrollable channels and a second plurality of selectively controllablechannels, where the first always-open channel and the first plurality ofselectively controllable channels are connected to a first arc fluidicpath, and the second always-open channel and the second plurality ofselectively controllable channels are connected to a second arc fluidicpath, where the first and second arc fluidic paths are arranged in acircle and not in fluid communication with each other, such that inoperation, the first always-open channel is selectively in fluidcommunication with one of the first plurality of selectivelycontrollable channels, while the second always-open channel isselectively in fluid communication with one of the second plurality ofselectively controllable channels.

In one embodiment, the RPPM further comprises an input channel and anoutput channel positioned under the actuator in relation to theplurality of equally spaced-apart openings such that when the actuatoris rotated, a fluid is pumped from the input channel to the outputchannel.

In one embodiment, each of the RPVs and the RPPMs further comprises amotor for rotating the actuator incrementally by the angle θ. In oneembodiment, the motor comprises a spring-loaded tensioning motor head ora self-tensioning motor head, where the self-tensioning motor headcomprises a cylinder body, where the cylinder body has one or morehelically cut slits around an axis of the cylinder body, or two or morehorizontally cut slits alternatively in X and Y directions to allowtension to be applied in the direction of the axis of rotation of theRPPM or RPV.

In one embodiment, each of the one or more on-chip pumps comprises apneumatically actuated peristaltic pump. In another embodiment, each ofthe one or more on-chip pumps comprises a mechanically actuatedperistaltic pump.

Furthermore, the perfusion controller may have at least one bubble trapcoupled to the fluidic network for removing bubbles therefrom.

In one embodiment, the at least one bubble trap comprises first andsecond microfluidic channels located at different levels defining afluidic compartment therebetween, a vertical via for connecting thefirst and second microfluidic channels, a bubble withdrawal channelplaced over the vertical via, and a hydrophobic gas exchange membraneplaced between the via and the bubble withdrawal channel for separatingthe fluidic compartment from the bubble withdrawal channels. In oneembodiment, an optional bubble accumulation area can be placed above thevertical via with the gas exchange membrane as the chamber's ceiling.

In another embodiment, the at least one bubble trap comprises amicrofluidic channel containing a dense forest of micro-pillars withinthe fluidic path that act as bubble sieves catching passing bubbleswhile providing alternative parallel paths for fluid to move freelybeneath them, and a bubble accumulation chamber formed directly over themicro-pillars. In one embodiment, a ceiling of the bubble accumulationchamber is formed of a hydrophobic gas exchange membrane that allows forbubble removal either due to passive diffusion into the atmosphere ordue to actively applied gentle vacuum while preventing fluid escape.

In one embodiment, the plurality of bio-objects is connected to eachother through the fluid bus in series, parallel, or a combination ofthem. In one embodiment, the at least one bio-object is operablybypassable from the other of the plurality of bio-objects.

In one embodiment, the perfusion controller is formed integrally with anoptically transparent material.

In another aspect, the invention relates to a system for analysis of aplurality of bio-objects. The system in one embodiment includes anetwork of perfusion controllers having a plurality of perfusioncontrollers as disclosed above. The plurality of perfusion controllersis arranged in an array for perfusing the plurality of bio-objectsindividually or simultaneously.

In one embodiment, the plurality of perfusion controllers is arranged inseries, parallel, or a combination of them. In another embodiment,combinations of the plurality of bio-objects and the plurality ofperfusion controllers are themselves arranged in series, parallel, or acombination of them to form the network of perfusion controllers.

In yet another aspect, the invention relates to a method for analyzing aplurality of bio-objects. The method includes the steps of providing aplurality of fluids, providing a fluidic network configured to be influid communication with the plurality of bio-objects and the pluralityof fluids, where the fluidic network comprises a plurality of fluidicswitches, one or more on-chip pumps, and a plurality of fluidic pathsconnected therebetween, and controlling the plurality of fluidicswitches and the one or more on-chip pumps to selectively andindividually perfuse at least one of the plurality of bio-objects withat least one of the plurality of fluids at a predetermined perfusionflow rate and deliver an effluent of the at least one bio-objectresponsive to the perfusion to a predetermined outlet destination foranalysis, recirculation, waste exhaust, or input to other bio-objects ofthe plurality of bio-objects.

In one embodiment, the fluidic network further comprises a microclinicalanalyzer (also termed in some usages a microchemical analyzer).

In one embodiment, the method further comprises the step of calibratingthe microclinical analyzer.

In one embodiment, the method also includes the step of detectingproperties of the effluent of the at least one bio-object.

Additionally, the method further has the step of measuring a pressuredrop across the at least one bio-object perfused with the at least onefluid, so as to regulate the flow rate of the at least one fluid throughthe at least one bio-object at the predetermined perfusion rate assumingthe fluidic resistance of the bio-object is known or calculable.

Furthermore, the method may have the step of removing bubbles generatedin the fluidic network.

In a further aspect, the invention relates to a microclinical analyzerusable for analysis of one or more bio-objects. In one embodiment, themicroclinical analyzer comprises a fluidic network having a plurality offluidic switches, a plurality of fluidic paths in fluid communicationwith the plurality of fluidic switches, and one or more on-chip pumpscoupled to corresponding fluidic paths, a sensor array coupled to thefluidic network, and a perfusion controller coupled to the fluidicnetwork and adapted for perfusing a bio-object with a desired fluid andoutputting an effluent of the bio-object responsive to the perfusion,where the fluidic network is configured such that the effluent of thebio-object is operably and selectively deliverable to the sensor arrayfor detecting properties of the effluent, to a predetermined outlet ofthe fluidic network, or to one of the plurality of bio-objects.

In one embodiment, each fluidic switch comprises a valve having at leastone pole and a plurality of throws, where the at least one pole isoperably and selectively in fluid communication with one of theplurality of throws.

In one embodiment, the microclinical analyzer further includes acalibration reservoir having a plurality of containers for containing aplurality of fluids, respectively, where the plurality of containers iscoupled to the plurality of throws of one of the plurality of fluidicswitches for individually providing the plurality of fluids to thesensor array for calibration. This calibration could be performed atrepeated intervals selected to track the biological activity of thebio-object. After each calibration operation, the sensor could bemaintained passively in one of the calibration solutions to maintainsensor performance in the interval between measurements of biologicalactivity and calibrations.

In one embodiment, the plurality of fluidic switches comprises first,second, and third fluidic switches, where the first fluidic switchcomprises a one-pole and four-throw valve coupled to the calibrationreservoir, the second fluidic switch comprises a four-pole andthree-throw valve coupled to the first fluidic switch, the sensor array,and the perfusion controller, and the third fluidic switch comprises aone-pole and four-throw valve coupled to the on-chip pump and outletsand another bio-object, where the second and third fluidic switches arecoupled to each other through the on-chip pump.

In one embodiment, each fluidic switch comprises a RPV and each on-chippump comprises a RPPM, where each of the RPV and the RPPM comprises anactuator having a circular ball-bearing cage defining a plurality ofequally spaced-apart openings thereon, and a plurality of ballsaccommodated in the plurality of equally spaced-apart openings such thatat least one opening accommodates no ball, where each two adjacentopenings through the center of the circular ball-bearing cage define anangle θ=2π/K, K being the number of the plurality of equallyspaced-apart openings.

In one embodiment, the RPV further comprises a plurality of selectivelycontrollable channels positioned under the actuator in relation to theplurality of equally spaced-apart openings such that at least oneselectively controllable channel is positioned under the at least oneno-ball opening or under at least one no-ball location of the circularball-bearing cage so that a fluid flow is allowed through the at leastone selectively controllable channel, while the other selectivelycontrollable channels are respectively positioned under the openingshaving the ball bearings so that no fluid flows are allowed through theother selectively controllable channels, where when rotating theactuator by a desired angle of (k×θ), k being 1, 2, . . . K, the atleast one cage opening without a ball or the absence of a cage openingis selectively placed over a desired one of the selectively controllablechannels.

In one embodiment, the RPPM further comprises an input channel and anoutput channel positioned under the actuator in relation to theplurality of equally spaced-apart openings such that when the actuatoris rotated, a fluid flow is pumped from the input channel to the outputchannel.

In one embodiment, each of the RPV and the RPPM further comprises amotor for rotating the actuator incrementally by the angle θ. In oneembodiment, the motor comprises a spring-loaded tensioning motor head ora self-tensioning motor head.

In yet a further aspect, the invention relates to an integratedbio-object microfluidics chip. In one embodiment, the integratedbio-object microfluidics chip comprises a fluid network. The fluidnetwork comprises a plurality of inlets for providing a plurality offluids, a plurality of outlets, a bio-object chamber for accommodatingat least one bio-object, first and second fluidic switches, and a firstpump. The bio-object chamber, the first and second fluidic switches, andthe first pump are coupled to each other in series. The first fluidicswitch is further coupled to the plurality of inlets for selectivelyreceiving one of the plurality of fluids therefrom and routing thereceived fluid to the first pump that in turn pumps the received fluidto the bio-object chamber so as to perfuse the at least one bio-objecttherein. The second fluidic switch is further coupled to the pluralityof outlets for selectively delivering an effluent of the at least onebio-object responsive to the perfusion to a predetermined outlet, or tothe first fluidic switch for recirculation.

In one embodiment, the integrated bio-object microfluidics chip furtherincludes a bio-object loading port coupled to the bio-object chamber forloading the at least one bio-object.

In one embodiment, each fluidic switch comprises a valve having at leastone pole and a plurality of throws, where the at least one pole isselectively operable in fluid communication with one of the plurality ofthrows.

In one embodiment, each fluidic switch comprises a RPV and the firstpump comprises a RPPM, where each RPV and RPPM comprises an actuatorhaving a circular ball-bearing cage defining a plurality of equallyspaced-apart openings thereon, and a plurality of balls accommodated inthe plurality of equally spaced-apart openings such that at least oneopening accommodates no ball bearing, where each of the two adjacentopenings through the center of the circular ball-bearing cage define anangle θ=2π/K, K being the number of the plurality of equallyspaced-apart openings.

In addition, the RPV further comprises a plurality of selectivelycontrollable channels positioned under the actuator in relation to theplurality of equally spaced-apart openings such that at least oneselectively controllable channel is positioned under the ball bearing,and at least one no-ball-bearing channel is open such that a fluid flowis allowed through the at least one selectively controllable channel,while the other selectively controllable channels are respectivelyclosed so that no fluid flows are allowed through the other selectivelycontrollable channels. As such, when rotating the actuator by a desiredangle of (k×θ), k being 1, 2, . . . K, the at least one no-ball openingor no-ball location in the bearing cage is selectively placed over adesired selectively controllable channels.

In one embodiment, the RPV also comprises at least one always-openchannel positioned under the actuator in offset from the plurality ofequally spaced-apart openings, such that the at least one offset channelis in fluid communication with the at least one selectively controllablechannel under the at least one no-ball opening or under at least oneno-ball location in the bearing cage, and the other selectivelycontrollable channels under the openings having the balls in the bearingcage are closed.

In one embodiment, each of the at least one always-open channel and theplurality of selectively controllable channels has an end connected toan arc fluidic path or a circular fluidic path.

In one embodiment, the at least one always-open channel has first andsecond always-open channels positioned under the actuator in offset fromthe plurality of equally spaced-apart openings, where the plurality ofselectively controllable channels comprise a first plurality ofselectively controllable channels and a second plurality of selectivelycontrollable channels, where the first always-open channel and the firstplurality of selectively controllable channels are connected to a firstarc fluidic path, and the second always-open channel and the secondplurality of selectively controllable channels are connected to a secondarc fluidic path, where the first and second arc fluidic paths arearranged in a circle and not in fluid communication with each other,such that in operation, the first always-open channel is selectively influid communication with one of the first plurality of selectivelycontrollable channels, while the second always-open channel isselectively in fluid communication with one of the second plurality ofselectively controllable channels.

In one embodiment, the RPPM further comprises an input channel and anoutput channel positioned under the actuator in relation to theplurality of equally spaced-apart openings such that when the actuatoris rotated, a fluid flow is pumped from the input channel directly tothe output channel for the purposes of continuously transporting fluidacross the device.

In one embodiment, each of the RPV and the RPPM further comprises amotor for rotating the actuator incrementally by the angle θ, where themotor comprises a spring-loaded tensioning motor head or aself-tensioning motor head.

In one embodiment the integrated bio-object microfluidics chip alsocomprises at least one bubble trap coupled to the fluidic network forremoving bubbles therefrom.

In one embodiment, the fluidic network further comprises a plurality ofcalibration solution ports for providing a plurality of calibrationsolutions for calibration, a third fluidic switch coupled to theplurality of calibration solution ports where the third fluidic switchis further coupled between the bio-object chamber and the second fluidicswitch, a second pump coupled to the third fluidic switch, and amicroclinical analyzer coupled between the second pump and the secondfluidic switch.

In one embodiment, the integrated bio-object microfluidics chip furtherincludes a chip carrier in which the fluidic network is formed.

Additionally, the integrated bio-object microfluidics chip also has amicrocontroller for controlling operations of the first and secondfluidic switches and the first pump. In one embodiment, themicrocontroller is provided with at least one of a wirelesscommunication protocol and a backup battery.

In one aspect, the invention relates to an integrated bio-objectmicrofluidics chip. In one embodiment, the integrated bio-objectmicrofluidics chip comprises first and second fluid networks. Each fluidnetwork comprises a plurality of inlets for providing a plurality offluids, a plurality of outlets, a bio-object chamber for accommodatingat least one bio-object, first and second fluidic switches, and a firstpump. The bio-object chamber, the first and second fluidic switches, andthe first pump are coupled to each other in series. The first fluidicswitch is further coupled to the plurality of inlets for selectivelyreceiving one of the plurality of fluids therefrom and routing thereceived fluid to the first pump that in turn pumps the received fluidto the bio-object chamber so as to perfuse the at least one bio-objecttherein. The second fluidic switch is further coupled to the pluralityof outlets for selectively delivering an effluent of the at least onebio-object responsive to the perfusion to a predetermined outlet, or tothe first fluidic switch for recirculation. The bio-object chambers ofthe first and second fluidic networks are substantially proximal to eachother while separated by a thin barrier or a membrane that allows forsignaling between the bio-object chambers of the first and secondfluidic networks.

In one embodiment, each of the first and second fluidic switchescomprises a RPV and the first pump comprises a RPPM.

In another aspect, the invention relates to an integrated bio-objectmicrofluidics chip. In one embodiment, the integrated bio-objectmicrofluidics chip has a fluidic network comprising a plurality ofinlets for providing a plurality of fluids, a plurality of outlets, abio-object chamber for accommodating at least one bio-object, aplurality of fluidic switches, and one or more pumps, where thebio-object chamber, the plurality of fluidic switches, and the one ormore pumps are coupled to each other such that at least one fluidicswitch operably and selectively receives one fluid from a correspondinginlet and routes the received fluid, through the one or more pumps, tothe bio-object chamber so as to perfuse the at least one bio-objecttherein, and one of the other fluidic switches operably and selectivelydelivers an effluent of the at least one bio-object responsive to theperfusion to a predetermined outlet, or to the at least one fluidicswitch for recirculation.

In one embodiment, each fluidic switch comprises a RPV and each pumpcomprises a RPPM.

In one embodiment, the integrated bio-object microfluidics chip furtherincludes a chip carrier in which the fluidic network is formed.

Further, the integrated bio-object microfluidics chip has amicrocontroller for controlling operations of the plurality of fluidicswitches and the one or more pumps. In one embodiment, themicrocontroller is provided with a wireless communication protocol and abackup battery.

In yet another aspect, the invention relates to an integrated bio-objectmicrofluidics chip cartridge. In one embodiment, the integratedbio-object microfluidics chip cartridge has a chip carrier, and at leastone integrated bio-object microfluidics chip including at least onefluidic network formed in the chip carrier. The at least one fluidicnetwork comprises a plurality of inlets for providing a plurality offluids, a plurality of outlets, a bio-object chamber for accommodatingat least one bio-object, a plurality of fluidic switches, and one ormore pumps. The bio-object chamber, the plurality of fluidic switches,and the one or more pumps are coupled to each other such that at leastone fluidic switch operably and selectively receives one fluid from acorresponding inlet and routes the received fluid, through the one ormore pumps, to the bio-object chamber so as to perfuse the at least onebio-object therein, and one of the other fluidic switches operably andselectively delivers an effluent of the at least one bio-objectresponsive to the perfusion to a predetermined outlet, or to the atleast one fluidic switch for recirculation.

In one embodiment, the integrated bio-object microfluidics chipcartridge further comprises a reservoir coupled to the plurality ofinlets for providing the plurality of fluids.

Additionally, the integrated bio-object microfluidics chip cartridgealso comprises a microclinical analyzer coupled to the fluidic networkfor detecting properties of effluent of the at least one bio-object.

Further, the integrated bio-object microfluidics chip cartridge has acalibration solution reservoir coupled to the microclinical analyzer forcalibration thereof.

Moreover, the integrated bio-object microfluidics chip cartridge mayfurther comprise a microcontroller for controlling operations of theplurality of fluidic switches and the one or more pumps of the fluidicnetwork and the microclinical analyzer, where the microcontroller isprovided with at least one of a wireless communication protocol and abackup battery.

In a further aspect, the invention relates to an integrated bio-objectmicrofluidics chip cartridge. In one embodiment, the integratedbio-object microfluidics chip cartridge has at least one inlet forindividually providing a plurality of fluids, at least one outlet, atleast one bio-object chamber coupled between the at least one inlet andthe at least one outlet, for accommodating at least one bio-object, atleast one perfusion control unit coupled to at least one bio-objectchamber for selectively perfusing the at least one bio-object with oneof the plurality of fluids, at least one microclinical analyzer coupledto the at least one perfusion control module for analyzing an effluentof the at least one bio-object responsive to the perfusion, amicrocontroller coupled to the at least one perfusion control module andthe at least one microclinical analyzer, and a chip carrier foraccommodating the at least one bio-object chamber, the at least oneperfusion control unit, at least one microclinical analyzer and themicrocontroller.

In one embodiment, the microcontroller is provided with at least one ofa wireless communication protocol and a backup battery.

In one embodiment, the carrier comprises a plurality of fluidic pathsformed therein for connecting the at least one inlet, the at least oneoutlet, the at least one bio-object chamber, the at least one perfusioncontrol unit, and at least one microclinical analyzer.

In one embodiment, the at least one perfusion control unit comprises atleast one fluidic network having a plurality of fluidic switches, andone or more pumps, configured such that at least one fluidic switchoperably and selectively receives one fluid from a corresponding inletand routes the received fluid, through the one or more pumps, to thebio-object chamber so as to perfuse the at least one bio-object therein,and one of the other fluidic switches operably and selectively deliversan effluent of the at least one bio-object responsive to the perfusionto a predetermined outlet destination, or to the at least one fluidicswitch for recirculation.

In one embodiment, the integrated bio-object microfluidics chipcartridge includes a mechanical controller for sensing strain andapplying either pneumatic or mechanical stresses to the at least onebio-object chamber.

Further, the integrated bio-object microfluidics chip cartridge includesa microscope or other means of optical imaging coupled to the at leastone bio-object chamber.

Additionally, the integrated bio-object microfluidics chip cartridgealso has a support system having at least one of a fluid unit coupled tothe at least one bio-object chamber and the at least one perfusioncontrol unit for providing the perfusion fluids, a gas supply unitcoupled to the at least one perfusion control unit, and a waste unitcoupled to the at least one perfusion control unit for exhausting theeffluent of the at least one bio-object.

Moreover, the integrated bio-object microfluidics chip cartridgeincludes a sample collection unit coupled to the at least one perfusioncontrol unit.

In one embodiment, the integrated bio-object microfluidics chipcartridge further has an environment control unit designed to provide anappropriate physiological environment to at least one bio-object.

In another embodiment, the integrated bio-object microfluidics chipcartridge also has at least two individual flow channels that connectwith the at least one perfusion control unit, where one of the at leasttwo individual flow channels is adapted for an efferent flow, while theother of the at least two individual flow channels is adapted for anafferent flow.

In yet a further aspect, the invention relates to a system for analysisof a plurality of bio-objects. The system in one embodiment includes aplurality of integrated bio-object microfluidics chip cartridges asclaimed above. The plurality of integrated bio-object microfluidics chipcartridges is arranged in an array for analysis of the plurality ofbio-objects individually or simultaneously.

In one embodiment, the plurality of integrated bio-object microfluidicschip cartridges is arranged in series, parallel, or a combination ofthem.

Also, the system may further include a system controller for controllingthe plurality of integrated bio-object microfluidics chip cartridges soas to selectively analyze one or more of the plurality of bio-objectsindividually or simultaneously.

In one aspect, the invention relates to a system for analysis of aplurality of bio-objects. In one embodiment, the system has at least oneperfusion controller, at least one microclinical analyzer, and acontroller in communication with the at least one perfusion controllerand the at least one microclinical analyzer for controlling operationsof the at least one perfusion controller and the at least onemicroclinical analyzer as to selectively analyze one or more of theplurality of bio-objects individually or simultaneously.

In addition, the at least one perfusion controller comprises at leastone fluidic network having a plurality of fluidic switches, one or morepumps, and a bio-object chamber, configured such that at least onefluidic switch operably and selectively receives one fluid and routesthe received fluid, through the one or more pumps, to the bio-objectchamber so as to perfuse at least one bio-object therein, and one of theother fluidic switches operably and selectively delivers an effluent ofthe at least one bio-object responsive to the perfusion to apredetermined outlet destination, or to the at least one fluidic switchfor recirculation.

In another aspect, the invention relates to a rotary caster actuatorusable for an RPV and an RPPM. In one embodiment, the rotary casteractuator has a shaft; and a ball or roller bearing assembly angularlymounted onto the shaft, wherein the ball or roller bearing assembly hasan outer rim configured such that when the shaft rotates, the outer rimof the ball or roller bearing assembly rolls along a circular path.

In one embodiment, the ball or roller bearing assembly comprises asocket ball bearing cage angularly mounted onto the shaft.

In another embodiment, the ball or roller bearing assembly furthercomprises a pressure transfer bearing; a pressure holding plate held inplace for transferring tensioning pressure via the pressure transferbearing to microfluidic channels thereunder.

In yet another embodiment, the ball or roller bearing assembly furthercomprises a rotary encoder for proving feedback indication of thebearing position, and an interface collar for providing the attachmentof the shaft to a motor.

In yet another aspect, the invention relates to a device usable for anRPV and an RPPM. In one embodiment, the device comprises a spring loadedpressure inverter assembly utilized to convert the downward pressureexerted by a ball or roller bearing rotary actuator assembly into anupward force capable of opening a normally closed microfluidic channel.

These and other aspects of the present invention will become apparentfrom the following description of the preferred embodiment taken inconjunction with the following drawings, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment.

FIG. 1 shows schematically single-cell dynamics and cell-cellcommunication in VIIBRE (Vanderbilt Institute for Integrative BiosystemsResearch and Education) Multitrap Nanophysiometers (MTNP). An example ofthe types of data that can be obtained using living cells inmicrofluidic chips, and microfluidic chips connected in series. Group1—MTNP design: (A) Single-trap schematic. (B) Mask layout. (C)Fluoroscein-filled microfluidic channels. (D) Trap SEM. Group 2—T-celldynamics: (E) Brightfield and fluorescence images of Jurkat cells withquantum-dot marker of IL2 receptor α. (F) Cellular kinetic activitymeasured by differential video microscopy, with summation ofdifferential images vs. time showing toxin effect. (G) Primary humanmature dendritic cells (DC) (red arrows 11) and naive CD4+T-cells (greenarrow 12) in a MTNP. (H) Time-series of brightfield and Ca²⁺ images. (I)Ca²⁺ response by a single, DC-stimulated T-cell, marked by arrows in (H)Inset: automated cluster analysis of 447 ionomycin-stimulated T-cells,identifying three populations that would not have been separated byFACS. Group 3—Dynamic apoptosis assay: (J) Normal and (K) chronicmyeloid leukemia (CML) CD34+ hematopoietic stem cells (HSC) exposed to150 nM dasatinib. In contrast to normal HSCs, the CMLs were drugresistant. Group 4—Non-contact paracrine signaling: (L) Daisy-chainedMTNPs with mature DCs upstream and naive T-cells downstream. M) Ca²⁺activation in downstream T-cells immediately after the two MTNPs wereconnected. (N) Time course of Ca²⁺ transients from 7 T-cells in (M)demonstrating non-contact paracrine signaling between DCs and T-cells[14, 15].

FIG. 2 shows schematically Omni-Omics: a monitoring, control, anddiagnostic system for a five-organ-on-a-chip system according to oneembodiment of the invention. Each highly instrumented microbioreactor(MBR) or Organ-on-a-Chip allows real-time image analysis and in-chamberfluorescent and electrochemical sensors that provide the controlalgorithm with dynamic output signals reporting the metabolic andmorphological state of the cells under study. A downstream ionmobility-mass spectrometer (IM-MS) provides near-real-time metabolomicand proteomic data from cellular secretions to characterize tissuephysiological state and response to drugs and toxins. On the order of ahundred computer-controlled microfluidic valves serve as actuators todeliver control inputs to specific MBRs to dynamically regulate theextracellular microenvironment. Micropumps and valves between chamberscontrol signaling between organs. Optogenetics could be used to extendthe control to inside the cell. The systems model would drive themultiple input-multiple output (MIMO) controls that close the loopbetween the sensors and the actuators, ultimately allowing control overdevelopment.

FIG. 3 shows schematically Ion Mobility-Mass Spectrometry (IM-MS). Thechemical communication between different organs in amulti-organ-on-a-chip system can be studied using IM-MS. (A) Theprinciple of an IM drift cell: a stack of ring electrodes creates anelectric field gradient that supports gas phase electrophoresis. (B) Ahypothetical IM separation for peptide ions exhibiting two distinctstructural sub-populations corresponding to globular (left) and tohelical (right) conformations. The observed arrival time distribution(ATD) data (top axis), can be transformed to a collision cross-sectionprofile (bottom axis). (C) IM-MS block diagram. (D) 3D MALDI-IM-MSconformation space plot for a complex protein digest, showingprojections of the mass spectrum without IM (back) and theelectropherogram (right) to the right with IM alone. (E) A theoreticaldepiction of where singly charged analytes of different molecularclasses are observed in IM-MS conformation space, providing the basisfor simultaneous omic experiments with minimal sample pretreatment andpurification [3-13].

FIG. 4 shows schematically a Microformulator for real-time preparationof Organ-on-Chip perfusion media. The real-time control of the chemicalmake-up of a micro-organ perfusion system requires the formulation ofvery small volumes of precise mixtures of custom perfusate prepared inreal time in response to the changing physiological states of organs inthe system. This system shows the pneumatically controlledmicroformulator. (A) AutoCAD layout of the microformulator based on thedesign of Hansen et al. [1, 2], but capable of assembling and mixing alarger volume of fluid. The device has 45 control channels, shown inred, and 16 fluid inputs, shown in blue. (B) Functional microformulator.(C) Valve actuation in the device. (D) Peristaltic pumping. (E) and (F)The custom solenoid valve banks.

FIG. 5 shows schematically a Rotary Planar Peristaltic Micropump (RPPM)and Rotary Planar Valve (RPV) according to embodiments of the invention.(A) A schematic of fluidic flow generated by rotatably moving the ballbearing 7 on the fluidic channel. (B) Actuators with ball bearings. (C)A schematic of a motor head with an actuator. (D) A schematic of a RPPM.(E) A stand-alone peristaltic pump having stepping motor andmicrocontroller controller and pump cartridge. (F) A miniature gearheadof the stepping motor connected to a miniature RPPM. (G) A schematic ofchannel layout for a RPV that selects one of four channels by a 15°rotation.

FIG. 6 shows schematically the RPPM-RPV batch-mode microformulator. Afourteen-port RPV1 (A) selects the reagent to be drawn from one of 14reservoirs by the RPPM (B) to load the shuttle (C). RPV2 (D) controlswashout, loading, mixing, and delivery of custom solutions.

FIG. 7 shows schematically the Omni-Omics automated biological explorer.A computer-controlled microformulator (A) prepares in one minute a μL ofcustom culture media delivered to cells (B) in a multitrapnanophysiometer (MTNP) on a fully automated inverted fluorescencemicroscope (C). The MTNP effluent is delivered to glucose, lactate,oxygen and pH electrodes (D) and to an automated pump (E) andaffinity-column desalter (F). The desalted media, which can upon commandinclude cellular lysate, is introduced by nanoelectrospray to an ionmobility-mass spectrometer (G). All instrument functions are under localcomputer control (H), and data are passed to a real-time SQL server (I).Computer algorithms (J) evaluate about 1000 candidate models and insilico experiments for each experiment design that is passed back to thereal-time SQL server (I) for implementation by the local computercontrol (H). The inferred model (K) is updated for immediate access byon-line investigators and for system control.

FIG. 8 shows schematically a perfusion controller according to oneembodiment of the invention. The exemplary perfusion controller canoperate in three modes: 1) Blood substitute bypasses Organ N while theinput to Organ N is connected to an on-chip fluid-selector valve thatallows the perfusion of the organ with a dye, drug, or media drawn fromthe fluid bus; 2) Organ N is between Organs N−1 and N+1 so that thethree organs are perfused in series; and 3) Organ N is removed from thesystem for a stop-flow measurement, repair, or replacement. In theconfiguration, Organ N Cartridge is disconnected from the system, andthe effluent from Organ N−1 goes to Organ N+1. A drug is injected onlyinto Organ N. Media can follow the drug to wash out unbound drug. Theeffluent is sent to waste so as not to expose other organs directly tothe drug. The two on-chip pressure sensors measure the absolute pressurein the organ inlet and outlet. Their difference reflects the pressuredrop across the organ, and thereby allows control of the speed of theon-chip pump to regulate system flow. The pump output can be eitherrecycled back into the organ, sent to the microclinical analyzer, towaste, or to Organ N+1. Additional features of this design can supportmulti-organ parallel perfusion connectivity by redefining the Media andWaste switch positions to correspond to Arterial Supply and VenousDrain, respectively. Therefore, this type of perfusion controller can beused for either series or parallel organ perfusion, or a combinationthereof.

In one embodiment, the pump is a pneumatically actuated peristalticpump, or a peristaltic pump with multiple, independent mechanicalactuators. Accordingly, application of no pressure leaves the pump inthe normally open mode, thereby allowing free flow through the channels.In one embodiment, rotary planar peristaltic micropumps are used, and ifit is desired to run the organ with flow driven solely by other organsor off-chip pumps, then it is necessary to add a mechanical retractor tothe drive balls, or insert a unidirectional flapper bypass valve or aselector bypass valve across the pump.

FIG. 9 shows schematically a valve design according to one embodiment ofthe invention, (A) a schematic layout of channels, and (B) a schematicof the valve assembly.

FIG. 10 shows schematically two channel layouts (A) and (B) of a valvedesign according to two embodiments of the invention, with the arcremoved in (A) to lower fluid dead volume.

FIG. 11 shows schematically a four-way double-pole double-throw valvewith positions illustrated in (B) and (C) according to one embodiment ofthe invention, (A) a schematic layout of channels.

FIG. 12 shows schematically a right, left, or through T-valveconfiguration according to one embodiment of the invention.

FIG. 13 shows schematically a double valve operated with a single ballcage (B) according to one embodiment of the invention, (A) a schematiclayout of channels.

FIG. 14 shows a self-tensioning motor head for supplying constant forceto the IOM Chip according to four embodiments (A)-(D) of the invention.

FIG. 15 shows schematically two views (A) and (B) of a RPPM having anembedded strain gauge in PDMS for observing the positions of the ballbearings according to one embodiment of the invention.

FIG. 16 shows various implementations (A)-(D) of a rotary planarperistaltic micropump according to embodiments of the invention.

FIG. 17 shows schematically a double channel pump-head according to oneembodiment of the invention.

FIG. 18 shows schematically a parallel capillary vascular bioreactorwith built-in in-line bubble trap according to one embodiment of theinvention.

FIG. 19 shows schematically four stand-alone microfluidic bubble traps(A), (B), and (C) utilizing straight channels and (D) with pillars andbubble accumulation chambers according to one embodiment of theinvention.

FIG. 20 shows a bubble trap implementation of a microfluidic bubble trapwith straight channels according to one embodiment of the invention.

FIG. 21 shows schematically a bubble trap for incorporation into aperfusion controller, a microclinical analyzer, or an organ chip,according to one embodiment of the invention.

FIG. 22 shows schematically an alternative configuration of amicrofluidic bubble trap according to one embodiment of the invention.(A) Block diagram of bioreactor containing a bubble trap. The lowerlayer contains a splitter to evenly distribute flow throughout thedevice and a forest of posts to arrest bubbles. Above the forest ofposts is an accumulation volume into which bubbles rise after beingtrapped. (B) Picture of fabricated cell culture device/bioreactorcontaining a bubble trap. (C) A composite image of the bubbles (out offocus) that have risen into the accumulation volume above the posts,with flow passing undisturbed underneath as indicated by the in-focuscomet trails of fluorescent beads suspended in fluid that is passingaround the posts. (D) A test of accumulation of bubbles in the bubbletrap by introducing a stream of bubbles with a bubble generator coupledto the bubble trap. Practical maximum volume of bubbles is approximately80% of the accumulation volume. If the accumulation volume reachescapacity, a separate channel (not shown) can be used to aspirate themfrom the trap.

FIG. 23 shows schematically a microclinical analyzer according to oneembodiment of the invention. There are three operational modes enabledby the four-pole three-throw valve in this system: (1) the Organ Outputpasses over the sensor array for electrochemical measurements ofmetabolites. (2) The Organ Output bypasses the sensor array, and thesensor array is isolated to prevent sensor fouling by proteins in theperfusate during those intervals of time for which the sensor array isnot in use. (3) The Organ Chip perfusate bypasses the sensor array,allowing the sensor array to be calibrated with three or morecalibration solutions or loaded with wash media by means of a one-polefour-throw valve, with the waste sent to drain to protect all organsfrom calibration fluids.

A one-pole four-throw valve allows the effluent from Organ N to passonto the perfusion bus for that organ, to be delivered to an externalsampler, or to Organ N+1. Additional poles on the switches would enableadditional modes.

FIG. 24 shows schematically VIIBRE multichannel multipotentiostats. Theelectronics in this instrument are connected to electrochemical sensorsin a microclinical analyzer and used to sense the metabolic state ofmultiple organs-on-chip according to one embodiment of the invention.

FIG. 25 shows schematically a sensor array chip for the microclinicalanalyzer. This figure shows sensors for six microclinical analyzers thateach have five platinum electrodes for Glucose, Oxygen, Lactate, pHsensors, and a counter electrode.

FIG. 26 shows schematically tissue/cellular studies in the multianalytemicrophysiometer.

FIG. 27 shows a schematic of fluidic routing in an IOM chip according toone embodiment of the invention. Areas 2780 and 2790 are off-chip stocksolution storage vials, and area 2700 is all part of the fabricated,disposable IOM chip.

FIG. 28 shows a schematic of a single organ IOM chip with one pump, twovalves, and a central area for the cells/organ according to oneembodiment of the invention. The IOM chip supports cell loading,reperfusion, and sample retention for analysis.

FIG. 29 shows schematically various valve configurations of Valve 1(A)-(C), and Valve 2 (D)-(F), of the signal organ IOM chip shown in FIG.28.

FIG. 30 shows schematically valve configurations when recirculating cellmedia (A) and applying media to bio-sample with overflow to waste (B) ofthe signal organ IOM chip shown in FIG. 28.

FIG. 31 shows a schematic of an organ IOM chip with a built-inmicroclinical analyzer according to one embodiment of the invention.Appropriate fluidic connections for calibration solutions,recirculation, and cellular efferent analysis are included in thisdesign.

FIG. 32 shows a schematic of a double organ IOM chip with two pumps,four valves, and a thin membrane (3230) separating the two organsaccording to one embodiment of the invention

FIG. 33 shows a theoretical embodiment of an intelligent chip carrier inSBS format according to one embodiment of the invention.

FIG. 34 shows an implementation of an intelligent chip carrier in SBSformat according to one embodiment of the invention.

FIG. 35 shows a theoretical embodiment of an IOM chip with supportingchip carrier/cartridge according to one embodiment of the invention.This exemplary IOM chip has 8 microfluidic pumps or valves andcontrolling electronic hardware, with the pumps or valves interfaced tothe IOM chip.

FIG. 36 shows a schematic of an intelligent chip carrier in SBS formatconfigured as a perfusion controller/microclinical analyzer (PC/μCA)according to one embodiment of the invention.

FIG. 37 shows a six-channel μCA screen-printed sensor array with acustom-manufactured fluidic housing.

FIG. 38 shows schematically an Organ Cartridge that does not require themicrofluidic microvasculature or the microfluidic interstitial spaceaccording to one embodiment of the invention. This example illustratesan arrangement of subassemblies, including a mechanical controller thatsenses strain and applies either pneumatic or mechanical stresses to theorgan chip. The perfusion controller (PC) contains the pumps, pressuresensors, and microfluidics for perfusion, sample collection, drugdelivery, and waste disposal. The microclinical analyzer (μCA) uses acommercial, low-cost, screen-printed electrochemical electrode array tomake regular measurements of glucose, lactate, pH, and oxygen to trackorgan metabolic activity and health. For systems with two compartments,e.g., a tissue interstitium and a microvasculature or a bronchial spaceand a microvascular space, there will be parallel PCs and μCAs for eachcompartment. The fluid bus contains both the arterial and venous systemsand other fluids, e.g., nutrients, drugs. The gas supply will deliverO₂, N₂, CO₂, etc. The small connecting tubing will in fact be in theform of custom interconnects. Electrical wiring uses a cartridgeelectrical bus that connects to a multi-organ experimental platform. Theconnection could also be done with a wireless communication protocol.

FIG. 39 shows a schematic diagram of an organ cartridge according to oneembodiment of the invention. The cartridge contains a MechanicalController (MC) that senses strain and applies either pneumatic ormechanical stresses to the Organ Chip (OC). The perfusion controller(PC) contains the pumps, pressure sensors, and microfluidics forperfusion, sample collection, drug delivery, and waste disposal. Themicroclinical analyzer (μCA) can utilize a commercial, low-cost,screen-printed electrochemical electrode array or other type ofelectrode array, connected to a multichannel multipotentiostat to makeregular measurements of glucose, lactate, pH, and oxygen to track organmetabolic activity and health. For systems with two compartments, e.g.,an interstitium and a microvascular space or a bronchial space and amicrovascular space, there will be parallel PCs and μCAs for eachcompartment. The microvasculature and microfluidic interstitium aredisposable interconnects that may incorporate the discrete tubing showninterconnecting the various components. The underlying support systemsinclude a Master Electronic System Controller or microcontroller, aMicroscope, a Fluid Supply for nutrients, drugs, etc., a Gas Supply forO₂, N₂, CO₂, etc., a Waste line, a means for Sample Collection, and anEnvironmental Controller that adjusts O₂, CO₂, humidity, temperature,etc.

FIG. 40 shows a schematic diagram of an array of Organ Cartridgesaccording to one embodiment of the invention. The individual organcartridges can be connected together to form a multi-organ system inwhich fluids from one Organ Cartridge can be connected to other OrganCartridges. In this example, the Heart module is shown as deliveringfluids to the Lung module and the Liver module. The Lung and Liver areshown to be connected via a separate fluid connection. The underlyingsupport network supplies fluids, gases, and waste removal according tothe programmed status demands of each individual organ's PerfusionControl subsystem.

FIG. 41 shows schematically a parallel configuration of Organs-on-a-Chipaccording to one embodiment of the invention. The input to eachorgan-on-chip is connected to the common “arterial” supply line, and theeffluent from each organ is connected to the common “venous” line. Eachorgan chip as shown could also include an individual perfusioncontroller and microclinical analyzer, with the pumps being either inseries with the organ or in parallel. One or more vascular return pumps,with pressure regulators, return the venous flow to the arterialcirculation, with gas exchange being provided by either a discretemembrane gas exchanger or another such device, or through the gaspermeable properties of the material out of which the microfluidicdevice is constructed.

FIG. 42 shows schematically a parallel-series configuration ofOrgans-on-a-Chip according to one embodiment of the invention. The inputto each organ-on-chip is connected either to the common “arterial”supply line, and the effluent from each organ is connected to the common“venous” line, in parallel mode, or two or more organs can be connectedin series, here shown by the effluent from the gut entering the liverand then being passed to the common venous line. Each organ chip asshown could also include an individual perfusion controller andmicroclinical analyzer, with the pumps being either in series with theorgan or in parallel. One or more vascular return pumps, with pressureregulators, return the venous flow to the arterial circulation, with gasexchange being provided by either a discrete membrane gas exchanger oranother such device, or through the gas permeable properties of thematerial out of which the microfluidic device is constructed.

FIG. 43 shows schematically three perfusion controllers in an array ofOrgans-on-Chip according to one embodiment of the invention. The figureshows three different interconnected Organs. Upper: Introduction offluid in Organ N−1 with the effluent going to a Fluids-Out port foreither analysis or disposal. Middle: The cartridge for Organ N runningon internal recirculation with local gas exchange and internal shunt toregulate the flow. The Arterial circulation bypasses the cartridge tomaintain homeostasis of other organs. Lower: Organ N+1 is connectedbetween the arterial and venous circulations with the variable lowimpedance shunt regulating the organ flow for the given arterial-venouspressure difference. A sample is being withdrawn for external analysis.At the bottom of the figure, a pump provides venous return. Organ-levelgas exchangers or a master gas exchanger could be inserted if required.

FIG. 44A shows schematically a design of one particular microfluidiccompatible rotary planar device with design features that can be usedeither for use as a pump or as a valve. Two key advantages of thisdesign are: 1) the critical pre-use tensioning of the roller ballsagainst the flexible membrane is easily achieved by simply placing aknown weight or force against the rigid pressure holding plate; and 2)the ball bearing cage is implemented as ball containing sockets directlyand rigidly attached to the drive pin. For pre-tensioning, onceappropriate pressure has been added (possibly via a calibrated donutshaped weight) then simply tightening the holding screws will establisha known compressive force underneath the ball bearings to actuate thedesired pump or valve functionality. The pressure transfer bearinglocated under the pressure holding plate acts to enable low frictionrotation of the Teflon or other low-friction drive bearing while at thesame time providing uniform downward force pressure on the Teflon drivebearing. Since the shaft rotation is rigidly linked to the Teflon drivebearing, it allows for direct transfer of the rotation delivered eitherfrom a motor or a hand crank via interface collar to the fluid drivingball bearings. When the central shaft is rotated, typically via a motoror a hand crank, the rotary force is transferred to a Teflon or otherlow friction material which holds individual ball bearings captive inball cages. Alternatively shafted roller bearings could be used totransfer force into the deformable membrane. A rotary encoder assemblycan be used to provide electronic verification of ball speed and preciseball location—a critical parameter when the device is utilized as arotary planar valve assembly.

FIG. 44B shows bottom views of a drive bearing indicating that (a) ballsand (b) rollers are housed within the sockets, according two embodimentsof the invention. The drive bearing is utilized in the rotary planardevice shown in FIG. 44A.

FIG. 45 shows a variation of the pumping module shown in FIGS. 44A and44B where pumping channels are fabricated in hard plastics and coveredwith a flexible membrane forming one of the microfluidic channel sides.The membrane allows for channel closure when pressure is delivered by arolling ball bearing. The hard plastic fluidic channel can be fashionedwith semi-circular cross section to facilitate valve sealing.

FIG. 46 shows various implementations of an axle-driven,cam-follower-bearing type actuator used to implement the RPPMs and RPVsaccording embodiments of the invention, (A) with cam followersspaced-equally mounted onto the cam, (B) with one missing cam followerat a location; (C) with a position indicator at the location in whichthe cam follower is missed.

FIG. 47 shows various implementations of an axle-driven, roller-bearingtype actuator used to implement the RPPMs and RPVs according embodimentsof the invention, (A) with rollers mounted into the spaced-equallysockets, (B) with one roller missed in a socket; (C) with a positionindicator at the location in which the roller is missed.

FIG. 48 shows the design of a roller or ball bearing caster pumpassembly that can be used to create a peristaltic pump when used inconjunction with a planar microfluidic channel covered by a flexiblemembrane. The design incorporates a roller bearing mounted at anapproximate 45 degree angle on a motor driven shaft. As the motor shaftrotates, the portion of the roller bearing in contact with the planarflexible membrane will trace a circular path on top of the embeddedfluidic channel. Only one rounded edge of the roller or ball bearingouter rim will be in compressional contact with the flexible membrane,and the rolling rim bearing action will exert minimal frictional slidingforce on the flexible membrane, thus creating as a very efficient longlived pump. This approximate 45 degree rotary caster design can also beused to provide rotary actuation of planar valve assemblies. Animportant feature of this design is that rotary shaft encoders can beeasily attached to the rigid shaft coupling to provide exact informationas to which portion of the circular arc contact region is currentlycompressed—thus facilitating exact control over planar fluid switchconnection modes.

FIG. 49 shows the conceptual design of a spring loaded pressure inverterwhich is comprised of a rotary array of actuators that can be used toprovide a plurality of normally-closed fluidic channel connections whichare driven by a central motorized rotary device, such as thatillustrated in FIG. 46, or alternatively by any of the ball bearing cageRPV actuators described previously. The device operates on the basis ofan embedded or otherwise rigidly mounted fulcrum which can transform thedownward pressure associated with a tensioned ball bearing, or rollerbearing into an upward force that can open a normally closedmicrofluidic valve. In this conceptual visualization eight NormallyClosed (N.C.) microfluidic valves are located in the central region ofthe planar assembly.

FIG. 50 shows additional views of the conceptual design for a springloaded pressure inverter actuation device for opening normally closedmicrofluidic valves. Note that the illustrated conceptual compressionsprings provide a force which is translated via a fulcrum mounted leverto provide the downward force that keeps a microfluidic channel closed.Only when external force is applied to a lever by a rotary actuatorelement will the associated microfluidic valve location be opened. Noteespecially that this is a conceptual diagram, and that actual physicalimplementation of the assembly might utilize flexible elastomers toprovide spring forces and one-piece integrated fulcrum flexure units toact as levers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which exemplary embodimentsof the invention are shown. This invention may, however, be embodied inmany different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art. Likereference numerals refer to like elements throughout.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term are the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatthe same thing can be said in more than one way. Consequently,alternative language and synonyms may be used for any one or more of theterms discussed herein, nor is any special significance to be placedupon whether or not a term is elaborated or discussed herein. Synonymsfor certain terms are provided. A recital of one or more synonyms doesnot exclude the use of other synonyms. The use of examples anywhere inthis specification including examples of any terms discussed herein isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

It will be understood that when an element is referred to as being “on”another element, it can be directly on the other element or interveningelements may be present therebetween. In contrast, when an element isreferred to as being “directly on” another element, there are nointervening elements present. As used herein, the term “and/or” includesany and all combinations of one or more of the associated listed items.

It will be understood that, although the terms first, second, third,etc. may be used herein to describe various elements, components,regions, layers and/or sections, these elements, components, regions,layers and/or sections should not be limited by these terms. These termsare only used to distinguish one element, component, region, layer orsection from another element, component, region, layer or section. Thus,a first element, component, region, layer or section discussed belowcould be termed a second element, component, region, layer or sectionwithout departing from the teachings of the present invention.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising”, or “includes” and/or “including” or “has” and/or“having” when used in this specification specify the presence of statedfeatures, regions, integers, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, regions, integers, steps, operations, elements,components, and/or groups thereof.

Furthermore, relative terms, such as “lower” or “bottom” and “upper” or“top”, may be used herein to describe one element's relationship toanother element as illustrated in the Figures. It will be understoodthat relative terms are intended to encompass different orientations ofthe device in addition to the orientation depicted in the Figures. Forexample, if the device in one of the figures is turned over, elementsdescribed as being on the “lower” side of other elements would then beoriented on “upper” sides of the other elements. The exemplary term“lower” can, therefore, encompass both an orientation of “lower” and“upper”, depending on the particular orientation of the figure.Similarly, if the device in one of the figures is turned over, elementsdescribed as “below” or “beneath” other elements would then be oriented“above” the other elements. The exemplary terms “below” or “beneath”can, therefore, encompass both an orientation of above and below.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

As used herein, “around”, “about”, “substantially” or “approximately”shall generally mean within 20 percent, preferably within 10 percent,and more preferably within 5 percent of a given value or range.Numerical quantities given herein are approximate, meaning that the term“around”, “about”, “substantially” or “approximately” can be inferred ifnot expressly stated.

As used herein, the terms “comprise” or “comprising”, “include” or“including”, “carry” or “carrying”, “has/have” or “having”, “contain” or“containing”, “involve” or “involving” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to.

As used herein, the phrase “at least one of A, B, and C” should beconstrued to mean a logical (A or B or C), using a non-exclusive logicalOR. It should be understood that one or more steps within a method maybe executed in different order (or concurrently) without altering theprinciples of the invention.

As used herein, the terms, “microclinical analyzer”, “microchemicalanalyzer”, and its abbreviation “μCA” are exchangeable.

The description is now made as to the embodiments of the presentinvention in conjunction with the accompanying drawings. In accordancewith the purposes of this invention, as embodied and broadly describedherein, this invention relates to perfusion controllers, microclinicalanalyzers, integrated bio-object microfluidics chips and systemsutilizing the perfusion controllers and the microclinical analyzers andapplications of the same.

It is naïve to assume that a collection of organs-on-a-chip will existin a stable equilibrium. Excess metabolic activity of one region withoutconcomitant increases in oxygenation and nutrients will lead toacidification and/or unwanted downstream effects. In living systems,homeostasis is maintained by a plethora of chemical, neural, andbiomechanical signals. An organ-on-a-chip system will require anequivalent regulatory system.

The central hypothesis is that humoral factors are critical formaintaining the viability of each organ-on-a-chip. However, the vastmajority of the secreted factors and their actions on specific organsremain poorly understood to date.

FIG. 1 shows examples of how soft lithographic microfabrication can beused to create microfabricated devices to study immune cell function. Byutilizing this technology to create an artificial lymph node on a chip,it provides an unprecedented opportunity to maintain immune cells with aphysiologically realistic rate of media superfusion. The very smallvolume of the chamber ensures that the neurotransmitters, paracrine andautocrine factors, and metabolites are not diluted and can mimic invitro the humoral interactions that occur in the body. Extracellularmatrix (ECM) can be added to the chambers as required.

To understand multimodal chemical communication between organs, theinvention allows us to study the interactions of different organs as afunction of time. The uniqueness of the Omni-Omics approach is shown inFIG. 2. A series of interconnected bioreactors are instrumented andcontrolled to an unmatched level, providing over 100 simultaneousbiological readouts in near real time, and the ability to control, atthe same time, dozens of experimental parameters.

With this Omni-Omics system, among other things:

-   -   (1) the soluble and bound factors that are produced by the        individual organs-on-a-chip can be identified;    -   (2) the role of individual secreted molecules on organ function        can be understood;    -   (3) the transcriptomic, proteomic, and metabolic changes that        arise as a result of exposure to selected drugs and toxins can        be quantified;    -   (4) a high-throughput assay for screening therapeutic agents        against the insults listed under (3) can be developed; and    -   (5) the effects of targeted interventions across various in        vitro and in vivo transgenic models can be verified, which opens        the avenue for preclinical trials.

The power of this approach is the breadth of dynamic information that isobtained from organs-on-a-chip using mass spectrometric measurements ofsoluble and bound proteins and metabolites, electrochemical measurementsof metabolic activity, optical imaging of fluorescent reporters andcellular phenotype, and of course various omics assays, the mostimportant being transcriptomics, and the ability to control this systemwith advanced, machine-learning software.

According to the invention as shown in FIG. 2, one can createinterconnected, low-volume, microfabricated chambers for each organ;control the fluidic inputs into each chamber so that the outflow of onechamber is the input to another; use electrochemical sensors to measuremetabolic activity and state-of-the art mass spectrometry to identifythe molecular species in the fluid flowing between the various chambers;use real-time fluorescent imaging of all chambers to characterizecellular and molecular activities within each developing explant; and atthe end of each extended experiment conduct extensive characterizationof all of the tissues. The miniature pumps and valves disclosed in theinvention allow us to control drug and chemical delivery to eachchamber.

Bioreactors: Microfabricated bioreactors (MBRs) offer the unprecedentedopportunity to maintain tissue explants in a close-to-physiologicalenvironment [56], wherein the extracellular volume and fluidinterconnectivity between brain regions are sufficiently well controlledto study paracrine and autocrine signaling phenomena. We will usestandard microfabrication technologies for polydimethylsiloxane (PDMS)or other polymers or materials to construct an interconnected network offive or more bioreactors, each of which will maintain a small explantfrom the brain of a developing mouse. Each MBR will be designed to allowus to record hundreds of biological parameters from each brain region weare studying, including, but not limited to, those shown in FIG. 2.Cellular microbioreactors are routinely fabricated in the laboratory,and the existing designs can be readily adapted for the proposedexperiments with brain explants. Most important, the flow rates utilizedto maintain tissues in small MBRs are well matched to the flow rates ofnanoelectrospray, ion mobility-mass spectrometers (nESI-IM-MS) (100-500nL/min), so that we can sample the fluid being exchanged between organswith time resolutions of one minute. The low cost and small size ofthese reactors will let us study many MBRs in parallel using amicrofabricated multiplexer.

Analysis: The interconnecting chambers allow us to sample the humoralfactors with nESI-IM-MS. Beyond this, we also have the capability toexamine the molecular distributions within organs with a wide variety ofmethods (MALDI-IM-MS, transcriptome profiling, etc.). Selected reactionmonitoring (SRM) with mass spectrometry will allow us to track thechanges of molecules that demonstrate the most differential activitybetween the five regions studied. Other methods of analysis are depictedin FIG. 2.

Ion Mobility-Mass Spectrometry (IM-MS): Many important questions aboutcell signaling, cell-matrix interactions, and metabolomics defy currentanalytical instrumentation and controls. Real-time measurement iscritical for biological system control. In particular, the simultaneoushigh-frequency dynamic measurements of protein expression and thegeneration of metabolites and other signaling molecules exceed currentcapabilities. For example, normal mass spectrometry (MS) of tissue orthe media from cell culture or tissue perfusion would detect a largenumber of isobaric species that could not be differentiated solely uponmass-to-charge ratio (m/z). High performance liquid chromatography(HPLC) can separate isobaric species through an interaction with achemically selective stationary phase tailored for particular analytes(e.g., hydrophobic or hydrophilic), but a single HPLC separation canrequire an hour or more, and this precludes real-time control of thesystem while probing metabolic dynamics.

The biggest problem with the application of mass spectrometry to a largecollection of organs-on-a-chip is that it takes 30 minutes to an hourfor a single HPLC run, depending upon the separation column utilized.This means that one can sample each organ two to four times a day in a10-organ system. If one wants to track four 10-organ experimentalplatforms, one needs to analyze 40 cuvettes a day and is able to readout each organ only once a day. For a one monthpharmacokinetics/pharmacodynamics run, one acquires a large number ofcuvettes, requiring a large number of LC columns and lots of MC time.

There are advantages to stop-flow collection of a single organ at a timeso that the metabolites and biomarkers being tracked are concentrated.Off-line analysis requires that the collection of larger volumes can bestored and handled (a microliter is a small amount of fluid in thebottom of a cuvette). This creates a growing backlog of cuvettes toanalyze, at 30-60 minutes each. The alternative enabled by theseinventions is to utilize a perfusion controller that controls externalsample collection directly into microfluidic tubing connected to a massspectrometer.

As a major innovation in mass spectrometry for understanding thephysiology of coupled biological systems, a collaboration between theWikswo and McLean groups at Vanderbilt, the Lipson group at Cornell, andVallabhajosyula at CFDRC is applying symbolic regression [1], machinelearning [2], electrochemical [3] and optical sensing, andnanoelectrospray and MALDI ion mobility-mass spectrometry [4-24] toinfer the equations underlying metabolic and signaling dynamics [25-29],ultimately to control biological systems [29, 30]. Ion Mobility-MassSpectrometry (IM-MS), the keystone technique in this omni-omics effort,utilizes a post-ionization gas-phase electrophoretic separation on thebasis of structure prior to m/z determination. This technology enablesthree-dimensional separations (analyte structure, mass-to-charge, andsignal intensity) to be completed on a timescale of milliseconds [4, 6,7, 31-38]. The details of the IM-MS approach are shown in FIG. 3.

Another reason for high-speed mass spectrometer analysis is to allowmultiplexing of the analysis of effluent from a large number ofdifferent organs individually sampled at a much lower rate. But toobtain speed, one needs to desalt without waiting for a standard LCseparation that does not require desalting. The invented perfusioncontroller and microclinical analyzer can readily include on theiroutputs modules that would provide on-line desalting, for example with aone-minute desalting process for a 100 nL sample feeding directly intothe nanoelectrospray (nESI) port on a Waters Ion Mobility-MassSpectrometer (IM-MS). The great feature of IM-MS is that the IMgas-phase electrophoresis structural separation accomplishes in 1 mswhat HPLC can do in 60 minutes and UPLC can do in 30. The bandwidthincrease of five orders of magnitude is reduced by a factor of about 50by the time required to desalt, but the advantage of this approach isthat the 1 minute desalting might allow one to look at every organ in a10 Cartridge Experimental System once every 10 minutes. Alternatively,rapid desalting might be possible by using a miniature microdialysisdesalter.

Advanced Microfluidic Systems for Cellular Control: According to theinvention, one can exploit the capabilities of microfluidic devices toprovide restricted dimensions to localize small cell populations; lowflow rates; a high ratio of cell volume to media volume; the ability torapidly change media; and proximity of in situ, in-line, and downstreamsensors. The development of compact, low-cost, and easy to usemicroformulators allows rapid changes to the relative concentrations ofup to 16 different chemical reagents in the media superfusing the cellsunder study.

Microformulators for MIMO Bioreactor Control: The ability of biologiststo control temporally the chemical and fluidic environment aroundadherent or suspended cells in vitro is limited by the availabletechnologies: bulk fluid changes, possibly including centrifugation andresuspension; peristaltic and syringe pumps; manual and multiheadpipettes; and fluid-handling robots. The speed with which each approachcan change the fluid environment is limited, and the requiredinstruments can be costly and quite bulky. Microfluidic devices haveproven useful for studies of chemotaxis and cell-cell interactions, butthese devices often utilize either multiple syringe pumps costing $2,000or more each [39-47], or on-chip peristaltic pumps that require precisemicrofabrication, multiple solenoid valves, and an external source ofpressurized gas at a cost of about $500 per pump [48-50]. Hencebiologists seldom have the ability to simultaneously control theconcentrations of multiple chemicals, a MIMO prerequisite. Hansen andQuake accomplished this with a microformulator for proteincrystallization, albeit with an 80 pL stroke volume, a five nL mixer anda ten nL/min flow rate [51-53]. FIG. 4 shows VIIBRE's implementation ofthis approach, but designed to have a 160 pL stroke, a 0.5 μL mixer, and0.5 μL/min flow rate—matched to typical VIIBRE microbioreactors and thenESI-IM-MS. However, chip and tubing complexity and size and thefabrication cost of the solenoid valve controller in FIG. 4F (about$6,000) limits the use of this technology.

The inventors introduced a new on-chip peristaltic pump that requirespurchase of only a miniature geared stepping motor (about $200) and acontroller (about $100) but was somewhat labor intensive to fabricate(about 1 person-hour pump) [54]. This led the inventors to invent anentirely new class of rotary, planar peristaltic micropumps (RPPMs), asshown in FIG. 5. At about $20 to $45 per channel, they cost at least anorder of magnitude less than commercial instruments used for precisetemporal control of biological fluids [55]. These are precise,easy-to-use pumps. The simple motor/thrust bearing geometry developedfor the RPPM also enables a new class of rotary planar valves (RPVs) asshown in FIG. 5, which cost $1 to fabricate and whose motor andcontroller cost about $90. One RPPM and two RPVs can be combined tocreate a simple microformulator, as shown in FIG. 6, for 1/20^(th) ofthe cost of the valve controller in FIG. 4F. This will deliver on-demandcustom solution mixtures as required for MIMO microenvironmentalcontrol.

Modeling and Control: Real-time control of a quantity requires itsmeasurement in real time. Measurement of acidification alone is notsufficient, and it is necessary to measure in addition glucose, oxygen,and lactate fluxes to understand core carbon metabolism. But organmetabolism also involves the secretion and consumption of a plethora ofother biological molecules. Current analytical capabilities cannotprovide the needed simultaneous measurements of protein expression,metabolites, and other signaling molecules generated by cells. IonMobility-Mass Spectrometry (IM-MS) offers the potential to be the nexttransformative systems biology technology. IM-MS yields five orders ofmagnitude increase in systems throughput over liquid-chromatography massspectrometry (LC-MS) by utilizing a post-ionization gas-phaseelectrophoresis separation on the basis of structure prior to m/zdetermination [4, 6, 7, 31-38]. Nano-electrospray ionization (nESI),matrix-assisted laser desorption ionization (MALDI), ultraperformanceliquid chromatography (UPLC), or gas chromatography (GC) provide IM-MSinputs.

The Omni-Omic Automated Biological Explorer, shown in FIG. 7, isproviding us with unprecedented ability to study cellular signaling andmetabolism of small cell populations, and is moving towardscomputer-guided, closed-loop control of the experiments. The inventorshave already demonstrated in silico that the exploration/estimation andsymbolic regression algorithms can infer metabolic models with no apriori information [29]. Using this approach to develop the equationsfor real-time control of the microbioreactors, and eventually,analytical models, will allow us to alter the cellular microenvironmentsto positively affect neural development. This system is ideallyconfigured as a diagnostic platform for organ-on-a-chip assaydevelopment. Most important, this system is capable of being runremotely, i.e., of laboratory telescience that will allow toxicologistsat one location to conduct diagnostic tests on bioreactors at another.

For the purpose of this invention of a perfusion controller and amicroclinical analyzer, there are a number of basic module definitionsrequired to understand the central role played by both the perfusioncontroller and the microclinical analyzer for culturing, maintaining,and studying a collection of interconnected organs-on-a-chip.

Organ-on-Chip Module (OoC): Each synthetic organ needs to be housed in adisposable microfluidic cell growth and incubation chamber. Thesechambers can vary in design to accommodate the particular needs of thecells comprising the organ, but the overall footprint of eachOrgan-on-Chip will be standardized to accommodate insertion into thevarious support and interrogation modules which allow multi-organ drugand pathogen interactions to be studied.

Organ Cartridge for cellular Instrumentation and Support: Thesecartridges could have a standardized overall footprint, but containcustomized support microfluidics, pumps, electronics, valving andinstrumentation modules appropriate to each individual organ type. Eachcartridge is designed to accept a disposable Organ-on-Chip module whichhas been pre-conditioned in an Organ Farm or Organ Incubator and eachcartridge has provisions for easily inserting the module in a sterilemanner. In a similar manner, these standardized Instrumentation/Supportcartridges are designed to accommodate sterile insertion of the OrganCartridge into a larger assembly known as the Multi-Organ ExperimentalPlatform or Cartridge Dock which supports many individual OrganCartridges and provides the environment for multi-organ experiments.

Cartridge Dock: This is a multi-unit support module which can be used toprovide control of temperature and continuous-flow nutrient supply tomany individual disposable Organ-on-Chip modules housed within theirrespective Organ Cartridges. It will be used to provide the appropriategrowth sequences necessary to generate the mature biological tissuearrays to mimic individual organ types. The Cartridge Dock and/or theOrgan Cartridge provides facilities for initial loading of cells intopre-sterilized Organ-on-Chip modules and includes provisions forinserting or disconnecting one module without compromising sterility ofadjacent modules. This system has a standardized interconnect systemwhich can accommodate the most complicated as well as the leastcomplicated Organ Cartridge module. The Cartridge Dock is controlled bya stand-alone computer-based control system that provides organ-specificflow of nutrients, appropriate valving, and recording functions thatidentify the history of individual Organ-on-Chip modules. The CartridgeDock is stored in an incubator or Organ Farm, which provides the desiredambient temperature and humidity. A master experiment control computerprovides the control signals that establish valve and pump controlconditions to fluidically connect individual Organ Cartridges to otherOrgan Cartridges within the Cartridge Dock and maintain physiologicalhealth of the tissues. In addition the Master Control Computer (MCC) isresponsible for periodic calibration of the electrochemical sensorarrays and controls the valving and pumping operations which arerequired to perform electrochemical measurements, and to dispense fluidsfor external analysis.

Master Control Computer: This is the dedicated system which controls alloperational aspects of the Multi-Organ Experimental Platform. In aninitial, exemplary implementation, this system operates as a simpleinterpreter that performs sequential operations of valve activations,pump parameter activations, electrochemical sensor calibration, andmeasurement sequences and fluid dispensing and drug injection sequencesaccording to a pre-set protocol list of operations loaded into theinstruction queue by the scientists designing the multi-organexperiment. During the experiment the MCC records detailed time-stampedconfirmation of each sequential activity and it acquires allexperimental measurement data from electrochemical sensors and from thecomputer actuated microscopes. The Master Control Computer may rely onsecondary microcontrollers to perform time critical or compute/bandwidthintense operations such as microscope camera focusing or high speedrepetitive microstepping operations. Advanced techniques of sensorfeedback controlled operation could be investigated at later stages ofinstrumentation development via dynamic modification of the experimentprotocol list. The master control computer can communicate withsecondary microcontrollers either by a hardwired connection or by meansof a digital wireless communications protocol.

Referring to FIGS. 38 and 39, two Organ Cartridge systems are shownaccording to certain embodiments of the invention. The invented organcartridge is used to control an Organ-on-a-Chip and illustrates thebasic relationships between the various subsystems that define the OrganCartridge system. The two Organ Cartridge systems are essentially thesame, except that the Organ Cartridge shown in FIG. 38 does not includethe microfluidic interstitial or microvasculature space. The OrganCartridge includes a mechanical controller (MC) that senses electrical,mechanical, or fluidic signals and applies either pneumatic ormechanical stresses to the Organ Chip (OC). The perfusion controller(PC) contains the pumps, pressure sensors and microfluidics forperfusion, sample collection, drug delivery, and waste disposal. TheMicroClinical analyzer (μCA) can utilize a commercial, low-cost,screen-printed electrochemical electrode array connected to amultichannel multipotentiostat to make regular measurements of glucose,lactate, pH, and oxygen to track organ metabolic activity and health.For systems with two compartments, e.g., an interstitium, there will beparallel PCs and μCAs for each compartment. The microvasculature andmicrofluidic interstitium are disposable interconnects that mayincorporate the discrete tubing shown interconnecting the variouscomponents. The underlying support systems include a Master ElectronicSystem Controller, a Microscope, a Fluid Supply for nutrients, drugs,etc., a Gas Supply for O₂, N₂, CO₂, etc., a Waste line, a means forSample Collection, and an Environmental Controller that adjusts O₂, CO₂,humidity, temperature, etc. The fluid bus contains both the arterial andvenous systems and other fluids, e.g., nutrients, drugs. The gas supplywill deliver O₂, N₂, CO₂, etc. The small connecting tubing will in factbe in the form of custom interconnects. Electrical wiring will use acartridge electrical bus that connects to the Multi-Organ ExperimentalPlatform.

Specifically, the integrated bio-object Organ Cartridge has at least onebio-object chamber for accommodating at least one bio-object, at leastone perfusion control unit coupled to at least one bio-object chamberfor selectively perfusing the at least one bio-object with one of theplurality of fluids, at least one microclinical analyzer coupled to theat least one perfusion control module for analyzing an effluent of theat least one bio-object responsive to the perfusion, a microcontrollercoupled to the at least one perfusion control module and the at leastone microclinical analyzer, and a chip carrier for accommodating the atleast one bio-object chamber, the at least one perfusion control unit,at least one microclinical analyzer and the microcontroller. The carriercomprises a plurality of fluidic paths for connecting the at least oneinlet, the at least one outlet, the at least one bio-object chamber, theat least one perfusion control unit, and at least one microclinicalanalyzer to define a fluidic network.

In one embodiment, the integrated bio-object chip Organ Cartridgeincludes a mechanical controller for sensing strain and applying eitherpneumatic or mechanical stresses to the at least one bio-object chamber.

Further, the integrated bio-object chip Organ Cartridge could in someembodiments include a microscope coupled to the at least one bio-objectchamber.

Additionally, the integrated bio-object chip Organ Cartridge also has asupport system having at least one fluid unit coupled to the at leastone bio-object chamber and the at least one perfusion control unit forproviding the perfusion fluids, a gas supply unit coupled to the atleast one perfusion control unit, and a waste unit coupled to the atleast one perfusion control unit for exhausting the effluent of the atleast one bio-object.

Moreover, the integrated bio-object chip cartridge includes a samplecollection unit coupled to the at least one perfusion control unit.

In one embodiment, the integrated bio-object chip cartridge further hasan environment control unit designed to provide an appropriatephysiological environment to at least one bio-object.

In another embodiment, the integrated bio-object chip Organ Cartridgealso has at least two individual flow channels that connect with the atleast one perfusion control unit, where one of the at least twoindividual flow channels is adapted for an efferent flow, while theother of the at least two individual flow channels is adapted for anafferent flow.

These examples highlight many of the absolutely essential topologicalfeatures of functional connectivity between subsystems required forsuccessful Organ Cartridge design. Several of the most important aspectsof the perfusion controller as it relates to the Organ Cartridge designapparent in this exemplary drawing are outlined below:

-   -   (1) The spatial arrangement of Organ Cartridge subsystems and        their controls must be compact.    -   (2) Fluidic paths must be carefully designed to provide        appropriate physiological tissue support functionality while at        the same time providing low dead volume connectivity so that the        built-in microclinical analyzer chip can detect target molecules        with maximum sensitivity.    -   (3) The microscope subsystem must have unhindered access to the        organ incubation chamber and a clear path must exist for        transillumination of the Organ Chip to allow label-free        microscopic observation of organ tissues.    -   (4) Mechanical control features must be tightly coupled to the        Organ Chip for those tissues which require such stimulation.    -   (5) The perfusion control system is central to the design and        must be able to provide all the necessary fluidic path        adjustments and volume modulations required to maintain tissue        viability and on-chip chemical concentration measurements.    -   (6) The fluidic support system must include provisions for        supplying appropriate fluids, gases, and waste solution pathways        required for long-term viability of organ tissues within the        Organ Chip supported by the Organ Cartridge. The disposable        Organ Chip unit must be co-engineered with the support system        modules to allow precision alignment for “plug-in” attachment.        The Organ Cartridge is also semi-disposable, but the Organ Chips        essentially must be disposable.

FIG. 40 shows a schematic diagram that illustrates several importantaspects of how different types of individual Organ Cartridges areinterconnected in a Cartridge Dock to provide a multi-organ system. Theindividual Organ Cartridges can be connected together to form amulti-organ system in which fluids from one Organ Cartridge can berouted to other Organ Cartridges. In this diagram the Heart module isshown delivering fluids to the Lung module and the Liver module. TheLung and Liver are shown to be connected via a separate fluidconnection. The underlying support network supplies fluids, gases andwaste removal according to the programmed status demands of eachindividual organ's Perfusion Control subsystem.

FIG. 41 shows schematically a parallel configuration ofOrgans-on-a-Chip. The input to each Organ-on-a-Chip is connected to thecommon “arterial” supply line, and the effluent from each organ isconnected to the common “venous” line. Each Organ Chip as shown would bemaintained by its respective Organ Cartridge which could also include anindividual perfusion controller and microclinical analyzer, with thepumps being either in series with the organ or in parallel. One or morevascular return pumps located in the Cartridge Dock, with respectivepressure regulators, route the venous flow to the arterial circulation .. . . Gas exchange is provided by either a discrete membrane gasexchanger or another such device, or through the gas permeableproperties of the material out of which the microfluidic Organ Chip isconstructed.

FIG. 42 shows schematically a parallel-series configuration ofOrgans-on-a-Chip. The input to each Organ Chip is connected either tothe common “Arterial” supply line, and the effluent from each organ isconnected to the common “Venous” line, in parallel mode, or two or moreorgans can be connected in series, here shown by the effluent from thegut entering the liver and then being passed to the common venous line.Each Organ Chip as shown could also include an individual perfusioncontroller and microclinical analyzer, with the pumps being either inseries with the organ or in parallel. One or more vascular return pumps,with pressure regulators, return the venous flow to the arterialcirculation, with gas exchange being provided by either a discretemembrane gas exchanger or another such device, or through the gaspermeable properties of the material out of which the microfluidicdevice is constructed.

FIG. 43 shows a schematic representation of three Organ Cartridges in aCartridge dock with their respective perfusion controllers. The figureshows three different interconnected Organ Chips. Upper: Introduction offluid in Organ N−1 with the effluent going to a Fluids-Out port foreither analysis or disposal. Middle: The cartridge for Organ N runningon internal recirculation with local gas exchange and an internal shuntto regulate the flow. The arterial circulation bypasses the OrganCartridge to maintain homeostasis of other organs. Lower: Organ N+1 isconnected between the arterial and venous circulations with thevariable, low impedance shunt regulating the organ flow for the givenarterial-venous pressure differential. A sample is being withdrawn forexternal analysis. At the bottom of the figure, a pump located on theCartridge Dock provides venous return. Organ-level gas exchangers or amaster gas exchanger could be inserted if required.

Of particular note are the parallel organ-support architecture and theversatility of inter-organ connectivity which is built into theprogrammable Perfusion Control subsection of each organ chip cartridge:

-   -   (1) Each Organ Chip location is provided with its own support        network. The organ support network (bottom layer on the drawing)        is of a uniform design, allowing any type of standardized organ        chip to plug into any position in the organ array.    -   (2) The precise fluidic connectivity between Organ Chips is        controlled by the Perfusion Control subsystem built into each        Organ Cartridge. In this particular design (FIG. 40), the Heart        Cartridge is shown providing fluid to both the Lung Cartridge        and the Liver Cartridge, and the Lung and the Liver are also        shown as connected.    -   (3) The important conserved design elements apparent in this        diagram are consistent modular design and versatile inter-organ        connectivity allowing for flexible multi-organ experiment        protocols.    -   (4) The details of the interconnects between organs, perfusion        controllers, the microfluidic vasculature, the microfluidic        interstitium, and the fluid, gas, and waste buses can be        configured to suit the specific needs of an individual        experiment and an Organ Chip array, either at the initiation of        the experiment or during its course.

FIGS. 8 and 27 illustrate more details of a Perfusion Control subsystembuilt into each Organ Cartridge as disclosed above. This exampleillustrates one particular implementation of fluidic switchinterconnectivity which would enable versatile multi-organinterconnectivity and measurement. In this schematic diagram the fluidicpathways are controlled by multi-position fluidic switches, which couldbe implemented using a variety of techniques.

Specifically, the perfusion controller has a plurality of inlets forproviding a plurality of fluids (851, 852 and 853 as shown in FIG. 8), aplurality of outlets, and a fluidic network coupled between theplurality of inlets and the plurality of outlets and being in fluidcommunication with the plurality of bio-objects. The fluidic networkcomprises a plurality of fluidic switches 810, 820, and 830 and one ormore on-chip pumps 840 adapted for selectively and individuallyperfusing at least one of the plurality of bio-objects with at least oneof the plurality of fluids at a predetermined perfusion flow rate anddelivering an effluent of the at least one bio-object responsive to theperfusion to a predetermined one of the plurality of outlets, where theplurality of outlets is coupled to at least one of an analyzer 862, awaste port 863, one of the plurality of bio-objects, and the fluidicswitch network.

In this exemplary embodiment, the perfusion controller further includesa fluid reservoir 850 having a plurality of containers (851, 852, and853) for containing the plurality of fluids, respectively. The pluralityof containers is coupled to the plurality of inlets for respectivelyproviding the plurality of fluids. The desired fluids contain a dye forlabeling selective areas within the bio-object 851, a drug 852, a medium853 or the like.

The perfusion controller may also have a microcontroller forindividually controlling the plurality of fluidic switches and the oneor more on-chip pumps of the fluidic network as so to control a flowrate of each fluidic path.

The perfusion controller further includes one or more sensors 870coupled to the at least one bio-object for measuring a pressure dropacross the at least one bio-object perfused with the at least one fluid,so as to regulate the flow rate of the at least one fluid through the atleast one bio-object at the predetermined perfusion rate, provided thebio-object resistance is known or calculable.

The fluidic network is formed with a plurality of fluidic paths in fluidcommunication with the plurality of fluidic switches and the one or moreon-chip pumps, where each bio-object is disposed in a correspondingfluidic path.

Each fluidic switch comprises a valve having at least one pole and twoor more throws, where the at least one pole is selectively operable influid communication with one of the two or more throws.

The fluidic network comprises first, second, and third fluidic switches810, 820, and 830 and an on-chip pump 840. The first fluidic switch(810) comprises a one-pole four-throw valve, the second fluidic switch(820) comprises a two-pole three-throw valve, and the third fluidicswitch (830) comprises a one-pole four-throw valve. In this embodiment,the plurality of bio-objects includes organ N−1, organ N, and organ N+1,where the organ N−1 is coupled to the second fluidic switch, the organ Nis coupled between the second fluidic switch and the on-chip pump thatis in turn coupled to the third fluidic switches, and the organ N+1 iscoupled to the second and third fluidic switches.

The example shown can operate in three modes: 1) Blood SubstituteBypasses Organ N while the input to Organ N is connected to an on-chipfluid-selector valve that allows perfusion of the organ with a dye,drug, or media drawn from the Fluid bus; 2) Organ N is between OrgansN−1 and N+1 so that the three organs are perfused in series; and 3)Organ N is removed from the system for a stop-flow measurement, repair,or replacement.

In the configuration shown, Organ Cartridge N is disconnected from thesystem, and the effluent from Organ N−1 goes to Organ N+1. Therefore adrug is injected only into Organ N. Media can follow to wash out unbounddrug. The effluent is sent to waste so as not to expose the downstreamorgans directly to the drug. The two on-chip pressure sensors measureabsolute pressure between the organ inlet and outlet. Their differencereflects the pressure drop across the organ, and thereby allows controlof the speed of the on-chip pump to regulate system flow. The pumpoutput can be either recycled back into the organ, sent to themicroclinical analyzer, to waste, or to Organ N+1. Additional featuresof this design can support multi-organ parallel perfusion connectivityby redefining the Media and Waste switch positions to correspond toArterial supply and Venous Drain, respectively. Hence this type ofcontroller can be used for either series or parallel organ perfusion. Inaddition, according to embodiments of the invention, the pump could beupstream or downstream of either the organ or the μCA electrode arrays.

If the pump is a pneumatically actuated peristaltic pump, or aperistaltic pump with multiple, independent mechanical actuators, thenapplication of no pressure leaves the pump in the open position, therebyallowing free flow through the channels. Were rotary planar peristalticmicropumps (RPPMs) used and one desired to run the organ with flowdriven solely by other organs or off-chip pumps, then it would benecessary to add a mechanical retractor to the drive balls, or insert aunidirectional flapper bypass valve or a selector bypass valve acrossthe pump.

The three independent switch arrays shown on this diagram would becontrolled by a computer to enable a wide variety of modal capabilitiesas detailed in the description of FIG. 40. Note that while the basicorgan-to-organ connection supported by this design is an in-line seriesconnection between physically adjacent modules, the switch arrays allowother configurations as well, including organ bypass, fluid flushing,and stop-flow organ effluent analysis using the microclinical analyzerchip.

This configuration enables either direct series perfusion of a set oforgans, or through the Organ Effluent buses (one for each organ),parallel perfusion of the organs, as desired. The one-pole four-throwvalves on the input and output sides of the organ can be replaced withvalves with additional ports for selection of the input and outputconnections of all organs in the system from a number of possible fluidstreams.

Among other things, an important feature of this design is that eachOrgan Chip can have a dedicated, in-series or in-parallel pump that candrive flow through an organ independent of the flow through otherorgans.

Each organ also is shown as having on-chip pressure sensors that areused to regulate the flow through the system and determine whether thefluidic resistance of the organ is within the desired range.

The output valve of the circuit can direct the flow to a microclinicalanalyzer.

This design and similar ones using the multi-position valve approachoffer an extremely versatile set of features spanning the range of bothserial and parallel connectivity. A number of specific features of thisapproach are of fundamental importance for enabling reliable long-termviability of all the organs in a multi-organ module and also forenabling well-controlled experimental paradigms which can providephysiologically relevant data concerning drug and pathogen multi-organresponses.

Note particularly the following specific points:

According to the invention, each organ can be in series or in parallelwith its own perfusion metering pump. This is extremely important inthat it allows for each specific organ to individually receive specificperfusion flow rates. This is of absolutely fundamental importance for:

-   -   (a) The detailed balancing of relative organ perfusion rates in        order to obtain physiologically relevant data.    -   (b) Providing scientists who design and analyze experiments run        in the multi-organ experimental module with the detailed        perfusion rate history associated with each organ that might        have participated in a multi-organ related cascade of events        resulting in particular drug or pathogen responses.    -   (c) Providing scientists with ability to simulate naturally        occurring increases or decreases in individual organ blood flow        that would occur during normal physiological stimulation or        during particular types of traumatic or pathological events        being studied.    -   (d) Limiting safe levels of blood pressure supplied to        particular organ modules that may not be able to tolerate a full        systemic pressure drop which exists in the full Arterial-Venous        differential pressure drop.    -   (e) Allowing intermittent or stop-flow conditions within a        particular organ module, which may be desirable for increasing        the concentration of organ metabolites prior to analysis with        the microclinical analyzer, and providing additional organ        residence time for bolus delivered drugs, or biomolecules        emanating from other organs.

In addition, according to the invention, this system provides versatileand re-configurable organ interconnection capability and organ effluentanalysis capability. For example,

-   -   (a) The organs can be perfused with fresh (non-recirculated)        media for external analysis.    -   (b) The organs can be connected in parallel, sharing common        recirculating Arterial and Venous fluidic paths, as depicted in        FIG. 43. A simple pressure-regulated pump circuit such as that        shown in FIG. 43 could recycle Venous System fluids to the        Arterial System supply line.    -   (c) Individual organs can be connected in series, so that the        effluent from one organ is routed into the fluidic input of        another organ, as shown in FIG. 8.    -   (d) An individual organ can be temporarily isolated from the        Arterial-Venous system and be reperfused with its own        recirculated effluent, as shown in FIG. 30A, for simulating        local oxygen starvation or poor organ blood flow, and/or for        allowing concentrations of metabolic products to increase prior        to analysis.    -   (e) A simple expansion of the number of switch positions        available on the fluid input and fluid output beyond the        four-position switches shown in this particular diagram, as        shown in FIG. 43 could easily enable more complicated chains of        organ perfusate interconnection if that should be required for        particular multi-organ experiments.

In any case, it is important to minimize the fluid volume of all valves,interconnects, and tubing, since this volume may be a significant factorin determining the required cell growth chamber volumes in order to keepdilution effects from rendering the entire assembly physiologicallyunrealistic.

An organ can be transiently placed in either a stop or localrecirculating mode to allow accumulation of paracrine signals andmetabolites prior to withdrawing a sample for external analysis. Bygiving each cartridge a pump and pressure sensors it is possible toensure the proper flow through each organ when operating in either theseries or parallel mode.

Chip oxygenation can be either through the PDMS or with Cartridge orsystemic gas exchange membranes.

According to the invention, the perfusion controller can also deliverfluid samples to the microclinical analyzer (μCA), which has threecomponents: the disposable microclinical analyzer chip which includesthe sensor array, the microfluidics, valves, and a pump, themicroclinical analyzer valve and pump drives which are implemented usingrotary motors or pneumatic controllers that attach to the disposable μCAchip, and the μCA Sensor Electronics which is shared between multiplemicroclinical analyzer units by using a microclinical analyzer SensorMultiplexer. This microclinical analyzer allows for the automatedimplementation of multianalyte microphysiometry, as invented by Cliffeland Wikswo, within a single Organ Cartridge.

There are a large number of commercial instruments for measuringbioreactor process variables, such as glucose, lactate, pH, and O₂.Clinical analyzers with disposable enzyme films, e.g., YSI, have beenoptimized for measuring electrolytes in mL volumes of blood plasma andhave detectors with large surface areas and hence large dead space.Simple glucose sensors abound, but are not readily interfaced and do nothave long-term stability or calibration. The Molecular DevicesCytosensor Microphysiometer, manufactured for 15 years beginning in theearly 1990's, could measure only pH, albeit with milli-pH sensitivity,15 second time response and several microliter volumes enclosing about10⁵ cells. Measuring only pH is insufficient to detect many metabolicchanges in cells, since multiple mechanisms lead to acidification.Eklund et al. added glucose, lactate, and O₂ sensors to the Cytosensorusing expensive commercial potentiostats, and then replaced thesepotentiostats with an economical, custom unit. However, none of thesesystems offers the level of calibration control required for long-termstudies with a single set of sensors.

Conventional acidification microphysiometry measures the pH changesproduced by the energy metabolism of approximately 300,000 live cells ina 3-microliter chamber. Cliffel et al. added multiple additionalelectrochemical sensors for metabolic analytes into this chamber to givea complete dynamical picture of the live cell physiology as aMultianalyte Microphysiometer. Their four-analyte system currentlymeasures extracellular glucose, oxygen, lactate, and pH within amicrofluidic chamber simultaneously on the minute timescale. They havealso added the ability to monitor extracellular calcium and dopaminelevels for primary neurons and neuronal-like cells, and insulin forpancreatic islets. Physiological measurements include dynamicmeasurements of basal metabolic rates in various media,agonist/antagonist competition studies, toxicology, and dose-responsecurves. Cellular activity, metabolic dynamics, and recovery after drugexposure will be monitored directly. By combining all of the informationcontained in the multianalyte “biosignature” metabolic activities ofeach organ-on-chip, including metabolic pathway shifting from aerobic toanaerobic metabolism, the depletion of internal energy stores, and thedynamic decoupling of metabolic parameters can be observed.

In the applications of multianalyte microphysiometry, the inventors haveexplored metabolic toxicology, metabolic activity of neurons undergoingstroke-like conditions, macrophage activation, pancreatic isletresponses, and cancer cell metabolism, as shown in FIG. 26, whichsummarizes the preliminary studies on the metabolic effects of theactivation of T-cells in the microfluidic chamber, as well as looking atthe metabolic processes involved in the oxidative burst response of RAWmacrophage. Further, we have also adapted the multianalytemicrophysiometers to measure insulin release to study the physiology andfunction of extracted primary pancreatic islets as a means of testingtheir viability before possible transplantation. Finally, we haveextended the multianalyte microphysiometry to measure the Warburg effectof aerobic glycolysis in cancer.

According to one embodiment of the invention, the disposablemicroclinical analyzer chip includes three well-established components:an electrode sensor array for each chamber, microfluidic chambers, andpumps with valves. The sensor array chips are based on screen printingelectrodes commercially fabricated to the specifications, as shown inFIG. 25. In one embodiment, these chips include five screen printedplatinum electrodes per array onto 6×9 cm ceramic substrates with sixarrays per substrate chip. Two substrate chips can then measure the fourdifferent analytes (Glu, Oxy, Lac, pH, etc.) in each of the twelvedifferent chambers simultaneously. For glucose and lactate sensing,chemical inkjet printing or other methods can be used to deposit theindividual enzyme layers necessary for the selectivity of each sensor.Also, for oxygen sensing, a Nafion coating or other membrane can beapplied to the sensor to prevent biofouling. For the pH sensor, apH-sensitive iridium oxide layer will be electrodeposited onto thosespecific sensor electrodes. Good laboratory practices and sterilizationprocedures will ensure the quality control of these sensors. Thesesensors will be calibrated before use in the microclinical analyzer toverify their quality.

The Vanderbilt custom-built multichannel multipotentiostat, as shown inFIG. 24, is used in conjunction with the on-chip microclinical analyzerelectrode sensor arrays to provide calibrated readouts of selectedanalyte concentrations. Two of these multichamber multipotentiostats arerequired for a 12 location measurement setup with 10 individual organcartridges and two sensor arrays devoted to measuring the main fluidiclines.

In addition, these individual sensor array chips can be fabricated foreasy insertion into the microfluidics stage of the microclinicalanalyzer, allowing for convenient replacement of sensor arrays asneeded. The sensor array chips can be reused by chemically removing andre-depositing the enzyme and iridium oxide layers. When plugged into themicroclinical analyzer, the microfluidic valves select between in-linemeasurements of the fluids leaving the organ chambers or sets ofcalibration solutions with different concentrations of each analyte toensure in situ accuracy of the sensor readouts. These valves alsocontrol output fluidic sampling for analysis by ion mobility-massspectrometry (IM-MS). The flow rates utilized to maintain the tissuesare well matched to the flow rates of nanoelectrospray, ionmobility-mass spectrometers (nESI-IM-MS) (100-500 nL/min), so that thefluid being exchanged between organs can be sampled with timeresolutions of one minute. Ion Mobility-Mass Spectrometry is thekeystone technique in this omni-omics advance developed in the McLeanlaboratory to rapidly analyze for lipids, carbohydrates, peptides, andnucleotides simultaneously. This technology enables three-dimensionalseparations, such as analyte structure, mass-to-charge and signalintensity, to be completed on a time-scale of milliseconds.

Possible improvements in the microclinical analyzer include the abilityto add other analyte sensors as desired, some of which could be specificfor each organ, and the ability to expand the number of analytes in eachchamber with custom designs for the sensor arrays and improvedmultipotentiostats.

Referring to FIG. 23, a microclinical analyzer is shown according to oneembodiment of the invention, which illustrates some of the criticalfeatures which must be included in the microclinical analyzer subsystem.These are required in order to provide periodic calibration of theelectrochemical sensor elements used to assay the concentration ofbiomolecules in the organ effluent stream. This valve system operatesunder computer-directed control, such as processor, microcontroller, orthe like, to enable periodic multianalyte electrochemical measurementsfrom the upstream organ between time slots devoted to sensor cleansingand calibration which are necessary to ensure accurate measurements.

Specifically, the microclinical analyzer includes a fluidic networkhaving a plurality of fluidic switches 2310, 2320, 2330, a plurality offluidic paths in fluid communication with the plurality of fluidicswitches, and one or more on-chip pumps 2340 coupled to correspondingfluidic paths, a sensor array 2370 coupled to the fluidic network; and amicrocontroller (not shown) for individually controlling the pluralityof fluidic switches 2310, 2320, 2330 and the one or more on-chip pumps2340 of the fluidic network as so to operably and selectively deliver aneffluent of at least one bio-object to the sensor array for detectingproperties of the effluent, or to a predetermined outlet destination,wherein the effluent of the at least one bio-object is from perfusion ofthe at least one bio-object with a desired fluid performed within aperfusion controller 2380.

The microcontroller is provided with at least one of a wirelesscommunication protocol and a backup battery.

The microclinical analyzer, as shown in FIG. 23, also has a calibrationreservoir 2350 having four containers 2351, 2352, 2353 and 2354 forcontaining a plurality of fluids, respectively. In the embodiment, thefour containers 2351, 2352, 2353 and 2354 are coupled to the firstfluidic switch 2310 for individually providing the plurality of fluidsto the sensor array 2370 for calibration. Each fluidic switch is a valvehaving at least one pole and a plurality of throws, wherein the at leastone pole is operably and selectively in fluid communication with one ofthe plurality of throws. For example, the first fluidic switch 2310 hasone pole, PO, and four throws 1, 2, 3, and 4 respectively connected tofour containers 2351, 2352, 2353 and 2354. The second fluidic switch2320 has four poles PO1, PO2, PO3 and PO4 and three throws 1, 2 and 3.The third fluidic switch 2330 has one pole PO and four throws 1, 2, 3,and 4 respectively connected to Organ N Effluent Bus 2361, ExternalSampler 2362, Waste Port 2363 and Organ N+1. The pump 2340 is connectedto the third pole PO3 of the second fluidic switch 2320 and the pole POof the third fluidic switch 2330.

For such a microclinical analyzer as shown in FIG. 23, there are threeoperational modes enabled by the four-pole three-throw valve in thissystem: 1) the Organ Output passes over sensor array for electrochemicalmeasurements of metabolites. 2) The Organ Output bypasses the sensorarray, and the sensor array is isolated to prevent sensor fouling byproteins in perfusate. 3) The Organ Chip perfusate bypasses the sensorarray, allowing the sensor array to be calibrated with 3 or morecalibration solutions or loaded with wash media by means of a one-polefour-throw valve, with the waste sent to drain to protect all organsfrom calibration fluids. A one-pole four-throw valve allows the effluentfrom Organ N to pass onto the perfusion bus for that organ, to bedelivered to an external sampler, or to Organ N+1. Additional poles onthe switches would enable additional modes.

In one embodiment, the microclinical analyzer also delivers smallvolumes of organ effluent solutions for processing by an in-linedesalter connected to an Ion Mobility-Mass Spectrometer, or largervolumes to an Ultra-Performance Liquid Chromatography Mass SpectrometerSystem or other such mass spectrometer analytical approach.

Various perfusion controller switching array methods can be utilized inmulti-organ array systems. The most obvious implementation of coupled,multi-organ systems involves serial perfusion of the organs, with one ormore pumps in series, such that the effluent of one organ could be used,possibly diluted, as the input to a downstream organ. In these designscontrol features would rely on variable speed pumps and cartridgepressure sensors to monitor and control cross organ pressure drops. Asuperior and more physiological means is to use designs that involve aversatile parallel Arterial-Venous supply architecture which moreclosely mimics the normal physiological relationships between organs.Some of the initial versions of this type of configuration can be seenin FIGS. 41 and 42. Note that this general structure corresponds to whatwe term a “Parallel Organ Perfusion” network and that it isfundamentally different from the organs-in-series architecture. Innature, certain organs, such as Gut and Liver, do receive circulationmore akin to the organ-in-series structure depicted in FIG. 42.

FIG. 38 shows a more detailed schematic of a parallel-organ perfusioncontroller, which illustrates one possible implementation of a parallelmulti-organ perfusion network as an alternative to the parallelperfusion functionality which is inherent in the embodiment shown inFIG. 8. Note particularly the physiological relevance and experimentalversatility inherent in these schematic diagrams suitable for parallelperfusion of multi-organ experiment systems:

-   -   (1) Organs can be arranged between a parallel Arterial supply        and Venous return blood substitute recycling system which        mirrors normal mammalian physiology.    -   (2) Switching arrangements allow for normal flow or stop flow        measurement of chemicals in the organ efflux path.    -   (3) Alternative fluids (drugs or dyes) can be supplied        individually to any organ.    -   (4) Fluids can be collected for external analysis from the        effluent path of individual organs.    -   (5) Internal module pumps and variable fluidic resistance        pathways can be used to adjust the pressure across individual        organs and also to adjust the blood bypass ratio to establish        appropriate blood substitute distribution ratios between        individual organs.

Because the Multi-Organ Experimental Platform is intended to supportlong-term live tissue experiments, sterilization of the perfusioncontroller and the microclinical analyzer microfluidic chips must beconsidered during every stage of their design and manufacture. EthyleneOxide treatment will ensure sterility during shelf life but it isnecessary to ensure that the residual Ethylene Oxide has escaped fromthe plastic before it is in contact with fluid that perfuses livingcells. Alternative sterilization methods techniques include autoclaving,gas, alcohols, or radiation.

The sensors must be sterilely inserted even if the instrument is not ina sterile environment. The surrounding support instrument should haveatmospheric control with positive pressure to eliminate the inclusion ofcontaminated particles or spores in the cartridge loading area.

Microfluidic devices inherently constrain the atmosphere in and aroundthe experimental area. According to the invention, the characteristicsof the materials can be leveraged to enhance the control of theenvironment inside the materials instead of trying to control theexternal environment.

The temperature of the fluids moving through the perfusion controllercan be regulated either by regulating the temperature of the entireenclosing environment or by having temperature-control fluid or othermeans to ensure that the devices are at the desired temperature. Mostmicrofluidic devices are constructed from polymers which are excellentthermal insulators, allowing the control of temperatures inside thedevices without being concerned about accurate control of the externaltemperatures. Humidity can also be controlled inside the device byadding water jacket channels to keep water-permeable polymers saturatedin water, simulating an external atmosphere of 100% humidity. Inaddition, gases may be dissolved in the water jacket to control the gasatmosphere in the experimental areas.

Controlling only the internal environment simplifies the task ofmaintaining sterility by eliminating any air space that has highhumidity and therefore eliminating the promotion of fungal and bacterialgrowth that thrives in moist atmosphere.

Heated HEPA filtered air will create a dehydrating environment outsidethe fluidic device which will reduce fungal and bacterial growth anddestroy viral material faster than a standard atmosphere. This air mayalso be treated with such things as UV radiation and activated charcoalfilters to ensure sterility and control VOCs that may be present inlaboratories.

A disposable, adhesive-backed membrane that is permeable to oxygen,carbon dioxide, and water vapor can be used to eliminatecross-contamination or microbial infiltration from any ports that needto remain open to air when devices or subassemblies have to be handledoutside of a sterile, laminar flow hood.

Both the perfusion controller and the microclinical analyzer, as well asthe Organ Chip itself, require computer control to ensure long-term cellviability and maintenance of realistic conditions for physiologicalstudies. The computer control system must be capable of measuringimportant variables in the system, such as temperature, pressure,dissolved gases, nutrients, and metabolites, and responding accordingly.The software protocols are required for control of the multi-organsystem, including the Organ Chip Cartridge, the mechanical and perfusioncontrollers, and the microclinical analyzer, and the Support Systems,which include External Sample Perfusion using the valves and pumpsalready described. In one embodiment of this system, the control of allof these components is performed by the Master Control Computerdescribed above, or by a set of microcontrollers that in turn arecontrolled by the Master Control Computer, with connections eitherhardwired or wireless.

Microcontrollers, such as pump or valve controllers, or localtemperature, humidity, or gas composition controllers, orelectroanalytic modules, can be efficiently and economically integratedinto the system as local microcontrollers that operate under thedirection of the main Master Control Computer. This computer would beresponsible for all real-time control aspects of the growth of cells inthe Organ Chips and the subsequent experiments, and will also beresponsible for acquiring all relevant information from the varioussubsystem microcontrollers.

The Master Control Computer can implement a detailed protocol at presettime points and export the data as a time-stamped confirmation of eachindividual action performed and each individual measurement gatheredover the several week duration of the experiment. The database recordwill be crucial to understanding and interpreting the complicatedmulti-organ interactions, and it is very important that visualizationtools be created from the outset that will allow scientists to easilyvisualize multi-component graphs of data extracted from thecomprehensive database record.

The valves and pumps described in the invention can be implemented in avariety of ways, including pneumatic push-up or push-down, normally openvalves, membrane-between-glass microfluidic valves that are normallyclosed, electromechanically actuated valves, rotary planar valves androtary planar peristaltic micropumps, as shown in FIG. 5, or byvibrating membrane check valve pumps. The implementation of the designsshown here does not depend upon the specific type of valve being used.

As disclosed above, one aspect of the invention provides a new categoryof microscope-compatible devices that allow active control of cellculture parameters and fluid flows as needed for long-term culturing andanalysis of biological tissue and cellular constructs and assemblies.Although this invention is focused on the use of microfluidic chips forcell culture and thus terms the device a “chip carrier,” it is notrestricted to the application of the device to the control and perfusionof microfluidic systems. It could equally be applied to the manipulationof fluids in wells, small dishes, or other culture chambers.

The principal novel features of these devices, which are illustrated inthe accompanying FIGS. 28-36, are, among other things:

-   (1) A standardized external mechanical footprint that is compatible    with all microscope stages, for example using the industry-standard    Society of Biomolecular Screening (SBS) wellplate specification.-   (2) The chip carrier which can accommodate microfluidic bioreactors,    hollow fiber bioreactors, tissue constructs, tissue samples such as    those obtained from biopsies, and other types of cell growth    chambers. These types of biological sample-holders which reside    within the overall device are herein referred to as the “chip” and    the surrounding, supporting device is referred to as the “Chip    Carrier”.-   (3) In one embodiment, an optically transparent region within the    chip carrier which can accommodate glass or plastic slides of    various dimensions and microfluidic bioreactors, tissue constructs,    tissue samples such as biopsies, and other types of cell growth    chambers and thereby enable microscopic observation of the cells in    the device.-   (4) The Chip Carrier is designed to provide stand-alone fluidic    media and nutritional support for the biological material in the    chip. This is accomplished by including within the Chip Carrier    assembly one or more fluidic reservoirs and one or more controlled    fluid delivery pumps. The purpose of the pump is to provide a    continuous or intermittent delivery of cell growth media to the    biological material in the chip. In the simplest implementation the    chip carrier would include just one reservoir for recycled media.    Alternatively, it could contain two reservoirs, one for fresh cell    growth media, and another to contain the waste fluid which comes out    of the chip when the pump operates to introduce fresh cell growth    media into the chip. In the former case the media becomes    conditioned over time through the accumulation of cytokines,    metabolites, and signaling molecules, and the nutrient levels slowly    decline. In the latter case the media does not recirculate, the    concentrations of these products to which the cells are exposed do    not increase with time, and the concentrations of nutrients stay    constant.-   (5) In other embodiments of the Chip Carrier, multiple fluidic    reservoirs and either multiple pumps, or pumps used in conjunction    with fluidic input and/or output valves, could be used in order to    deliver various combinations of cell media, drugs, and other liquid    compounds to the biological material in the chip.-   (6) In other embodiments of the Chip Carrier, multiple fluidic    reservoirs and either multiple pumps, or pumps used in conjunction    with multi-position fluidic input valves could be used in order to    deliver various combinations of cell media, drugs, and other liquid    compounds to the biological material in the chip.-   (7) A key feature of the invented chip carrier is the inclusion of    an electronic microcontroller and an on-board battery. The purpose    of the microcontroller is to provide signals to the pump(s) and    fluidic valve(s) assemblies that feed the biological material in the    chip assembly. The purpose of the battery is to allow operation of    the pumps and valves in the carrier when it has been disconnected    from its standard power supply, for example during transport or    while being examined microscopically or chemically.-   (8) A key feature of the invented chip carrier is the ability of the    microcontroller to implement complicated fluid and drug delivery    protocols to the biological material on the chip. The protocols can    be preloaded into the microcontroller and the chip carrier can    implement these fluid delivery instructions without human    intervention.-   (9) The microcontroller can also make autonomous decisions regarding    pumps and valves based upon the output of embedded sensors.-   (10) Another feature of the invented chip carrier is that the    microcontroller can include a wireless electronic input port. This    input port can be used to deliver new fluidic control protocols to    the Chip Carrier microcontroller, and it can be used to temporarily    override existing protocols, for example: investigators may not want    fluid to flow through the chip assembly while they are observing the    biological sample on a microscope, or for example, investigators may    want to deliver certain fluids to the biological sample only when    the chip carrier is being observed on a microscope.-   (11) A key feature of one embodiment of the invented Chip Carrier is    the inclusion of on-board sensor elements (termed a microclinical    analyzer). This assembly of pumps and valves can be used to sample    the effluent from the biological material in the chip and use    electrochemical, optical, or other means to measure certain    metabolites or other specific molecules contained in the effluent    fluid. A key feature of the invented Chip Carrier design is the    inclusion of microprocessor-controlled pumps, valves, and    electrochemical sensor elements to accomplish this task. A key    feature of the invented Chip Carrier design is the ability of this    design to provide calibration and biofouling protection for the chip    carrier resident sensor elements.-   (12) A key feature of the invented Chip Carrier is that it can be    programmed to deliver precise amounts of cell chamber effluent on    demand to an external microfluidic port; for example, in situations    when investigators wish to use a device such as an external mass    spectrometer or other analytic instrument to analyze the output of    the chip.-   (13) One of the most important features of the invented Chip Carrier    is that it is a self-contained device which can be easily    transported between the long-term incubator environment and the    short-term microscope evaluation environment without compromising    sterility of the closed fluid delivery system and without    interrupting desired fluid delivery protocols.-   (14) A key feature of the invented Chip Carrier design is the closed    fluid delivery system which can accommodate pre-sterilized fluid    samples and pump/valve assemblies and which includes sterile vent    assemblies on all fluid supply and fluid waste assemblies. This is    extremely important in the context of long-term cell culture    experiments.-   (15) A key feature of the invented Chip Carrier design is the    ability of this design to accommodate in-line fluid de-bubbler    assemblies. These fluid de-bubbler assemblies are designed to    prevent small bubbles of air from interfering with cell assemblies    or fluid delivery channels.-   (16) A key feature of some implementations of the invented Chip    Carrier design is the inclusion of on-board micropumps to deliver    vacuum suction to operate certain classes of on-board debubbler    and/or mechanical attachments.-   (17) The on-board pumps could also provide computer-controlled    suction or pressure as required to operate various vacuum- or    suction-activated chips.-   (18) A key feature of some implementations of the invented Chip    Carrier design is the inclusion of on-board micropumps to deliver    pressurized sterile air to operate certain classes of on-board    oxygenator attachments.-   (19) The invented Chip Carrier contains a battery assembly which can    power all pumps and valves when the unit is disconnected from an    external charging station. The invented chip carrier design includes    a battery-charging circuit which allows batteries to be    recharged—typically during the periods when the chip carrier resides    within an incubator. The invented Chip Carrier design incorporates    an easy-insertion electrical connector for this purpose.-   (20) A key advantage of the invented Chip Carrier design is that the    fluidic system is completely enclosed. This offers the additional    advantage of allowing use in low humidity incubators, which are less    likely to corrode electrical contacts and less likely to support    fungal and bacterial contamination.-   (21) The invented Chip Carrier could also include on-board    temperature regulation, so that the chips could be maintained at a    desired operating temperature, either above or below the ambient    temperature, without the need for an external incubator.-   (22) The Chip Carrier can be implemented with one or more reservoirs    and one or more pumps but without a microcontroller.-   (23) The Chip Carrier can include fluid flow and fluid pressure    sensors.

Further aspects of the invention include, among other things, IntegratedOrgan Microfluidics (TOM) Chip and applications of the same.Particularly, various embodiments of the invention are focused on theintegration of pumps, valves, bubble traps, cell chambers and supportingfluid networks and interconnects into a single IOM Chip. The TOM can beused as one component of an organ-on-chip cartridge that includes, forexample, a chip carrier to support the IOM and the motors for pumps andvalves.

Conventionally, many Organ-on-Chip systems utilize discrete components,such as pumps and valves that are connected to the organ by tubing. Amajor problem of this approach is the volume of fluid contained by thetubing. Thus, it is important to minimize this volume and thereby avoidunnecessary dilution of metabolites and signaling molecules thatcomprise the chemical communication between different organs.

As disclosed in the invention, the IOM chip addresses this problem byintegrating on-chip rotary planar peristaltic micropumps (RPPMs), rotaryplanar valves (RPVs) and microfabricated bubble traps (MBT) with theorgan or organs on a single chip. In one embodiment, the TOM chip ismade out of an optically clear polymer to allow both fluid manipulationwithin the chip and light transmission for observation of biologicalsamples within the chip. This single, disposable chip sits in a “chipcarrier” as described previously which houses the stock solutions,driving electronics, and mechanical support for the insert. Thecombination of chip carrier and TOM comprises the organ-on-chipcartridge.

FIGS. 33-36 show various embodiments of an intelligent chip carrier, oran integrated bio-object microfluidics chip, according to the invention.

As shown in FIGS. 33 and 34, the integrated bio-object microfluidicschip has a base carrier 3310/3410 defining the footprint of the device,a plurality fluidic paths, holes and chambers/slides 3360/3460, etc. foraccommodating components of the chip. The components include, but arenot limited to, an inlet reservoir 3330/3430, an outlet reservoir3370/3470, a microfluidic device mounted to the chambers/slides3360/3460, a microcontroller 3340/3440 with wireless interface forin-incubator system pump control with one DC power connector, a backupbattery 3350/3450 for out-of-incubator transportation and tests, and aRPPM pump with a DC gearhead motor 3320. The base carrier is a plasticSBS-format chip carrier.

FIG. 35 shows another embodiment of the integrated bio-objectmicrofluidics chip, which has a cells/organ chamber 3570 foraccommodating cells/organ to be analyzed, reservoirs 3530, eightmicrofluidic pumps or valves 3520, and a microcontroller 3540 withwireless interface for in-incubator system pump control with a backupbattery for out-of-incubator transportation and tests. The base carrieris a plastic SBS-format chip carrier. FIG. 35 features an TOM chip,3570, in which all fluidic connections are made within the device,without the need for tubing to connect the microfluidic pumps and/orvalves.

In one embodiment, as shown in FIG. 36, the integrated bio-objectmicrofluidics chip, i.e., intelligent chip carrier, has at least oneperfusion controller (PC) 3650, at least one microclinical analyzer(μCA) 3620, and a microcontroller 3640 in communication with the atleast one perfusion controller 3650 and the at least one microclinicalanalyzer 3620. The integrated bio-object microfluidics chip cartridgealso has one or more of PC/μCA calibration solution vials 3632 and oneor more perfusion reservoirs and drug vials 3630 in communication withthe at least one perfusion controller 3650 and the at least onemicroclinical analyzer 3620. The integrated bio-object microfluidicschip cartridge in one embodiment may also have a power supply 3642.Further, the integrated bio-object microfluidics chip cartridge has abio-object chamber 3660. Additionally, the controller can be a wirelesscontroller.

In another embodiment, the integrated bio-object microfluidics chipincludes at least one fluidic network formed in the chip carrier. The atleast one fluidic network comprises a plurality of inlets for providinga plurality of fluids, a plurality of outlets, a bio-object chamber foraccommodating at least one bio-object, a plurality of fluidic switches,and one or more pumps. The bio-object chamber, the plurality of fluidicswitches, and the one or more pumps are coupled to each other such thatat least one fluidic switch operably and selectively receives one fluidfrom a corresponding inlet and routes the received fluid, through theone or more pumps, to the bio-object chamber so as to perfuse the atleast one bio-object therein, and one of the other fluidic switchesoperably and selectively delivers an effluent of the at least onebio-object responsive to the perfusion to a predetermined outlet, or tothe at least one fluidic switch for recirculation. In one embodiment,the at least one fluidic network defines the at least one perfusioncontroller (PC) 3650.

In an exemplary embodiment shown in FIG. 36 the integrated bio-objectmicrofluidics chip cartridge further comprises a reservoir 3631 coupledto the plurality of inlets for providing the plurality of fluids.

Additionally, the integrated bio-object microfluidics chip cartridgealso comprises a microclinical analyzer 3620 coupled to the fluidicnetwork for detecting properties of effluent of the at least onebio-object.

Further, the integrated bio-object microfluidics chip cartridge has acalibration solution reservoir 3632 coupled to the microclinicalanalyzer for calibration thereof.

Moreover, the integrated bio-object microfluidics chip cartridge mayfurther comprise a microcontroller 3640 for controlling operations ofthe plurality of fluidic switches and the one or more pumps of thefluidic network and the microclinical analyzer, where themicrocontroller is provided with at least one of a wirelesscommunication protocol and a backup battery 3642.

Referring to FIG. 27, a system for analysis of the bio-object includingan organ or a group of cells includes an integrated bio-objectmicrofluidics chip 2700 and a plurality of vials formed outside the chip2700 in areas 2780 and 2790. The vials are adapted for providing loadingof the cells, media, drugs and calibration solutions, and for outputtingwaste, etc. The integrated bio-object microfluidics chip or IntegratedOrgan Microfluidic chip (IOM) 2700 is formed with a fluid network havinga bio-object chamber 2760 for accommodating at least one bio-object,four fluidic switches/valves 2710, 2720, 2730 and 2740, two pumps 2751and 2752, and a microclinical analyzer 2770. The bio-object chamber2760, the first pump 2751, the first fluidic switch 2710, the secondfluidic switch 2720, the microclinical analyzer 2770, the fourth fluidicswitch 2740, the second pump 2752, and the third fluidic switch 2730 arecoupled to each other in series. The first fluidic switch 2710 isfurther coupled to the plurality of inlets/vials for selectivelyreceiving one of the plurality of fluids therefrom and routing thereceived fluid to the first pump that in turn pumps the received fluidto the bio-object chamber so as to perfuse the at least one bio-objecttherein. The effluent of the at least one bio-object responsive to theperfusion is then directed to the microclinical analyzer 2770 foranalysis. According to the embodiment, the microclinical analyzer 2770can be calibrated, as needed, by activating the third fluidic switch2730 that is further connected to the vials of calibration solutions,the second pump 2752 and the fourth fluidic switch 2740.

Referring to FIGS. 28-30, an integrated bio-object microfluidics chip2800 is shown according to another embodiment of the invention. Theintegrated bio-object microfluidics chip has a fluid network. The fluidnetwork comprises a plurality of inlets for providing a plurality offluids, such as media, drug 1, drug 2, a plurality of outlets, forexample, a waste outlet and an analysis outlet, a bio-object chamber2860 for accommodating at least one bio-object, first and second fluidicswitches Valve 1 and Valve 2, and a pump 1. The bio-object chamber, thefirst and second fluidic switches, and the first pump are coupled toeach other in series. The first fluidic switch is further coupled to theplurality of inlets for selectively receiving one of the plurality offluids therefrom and routing the received fluid to the first pump thatin turn pumps the received fluid to the bio-object chamber so as toperfuse the at least one bio-object therein. The second fluidic switchis further coupled to the plurality of outlets for selectivelydelivering an effluent of the at least one bio-object responsive to theperfusion to a predetermined outlet, or to the first fluidic switch forrecirculation. Each fluidic switch comprises a rotary planar valve (RPV)having a number of selectively controllable channels, for example, Valve1 has five channels 2821-2825, while Valve 2 has four channels2831-2834.

Additionally, the integrated bio-object microfluidics chip also has abio-object loading port coupled to the bio-object chamber for loadingthe at least one bio-object.

FIG. 29 shows channel selections by rotating the actuator 2811 and 2812to select the desired fluid to perfuse the cells/organ and selectivelyroute the effluent of the cells/organ responsive to the perfusion to apredetermined outlet destination. For example, as selected, the effluentof the cells/organ is recirculated in FIG. 30A, while exited to thewaste outlet in FIG. 30B.

FIG. 31 shows another embodiment of an integrated bio-objectmicrofluidics chip which includes a similar configuration. Additionally,the integrated bio-object microfluidics chip also includes a pluralityof calibration solution ports for providing a plurality of calibrationsolutions for calibration, a third fluidic switch coupled to theplurality of calibration solution ports, a second pump coupled to thethird fluidic switch, and a microclinical analyzer coupled between thesecond pump and the second fluidic switch. The third fluidic switch isfurther coupled between the bio-object chamber and the second fluidicswitch.

FIG. 32 shows yet another embodiment of an integrated bio-objectmicrofluidics chip which includes two fluidic networks 3210 and 3220,each of which is the same as that of the integrated bio-objectmicrofluidics chip shown in FIG. 28. The two fluidic networks 3210 and3220 are symmetrically formed in a chip carrier so that the bio-objectchambers of the first and second fluidic networks are proximal to eachother while separated by a thin barrier or a membrane 3230 that allowsfor signaling between the bio-object chambers of the first and secondfluidic networks.

According to embodiments of the invention, the IOM Chip is sterilizable,and multiple configurations of pumps and valves are created fordifferent experimental methodologies. Variations of this design are usedto (1) perfuse a single organ or group of cells, (2) perfuse two groupsof cells connected by a thin porous membrane or barrier, or (3) perfusean organ with an on-board clinical analysis system, e.g., microclinicalanalyzer. To support these end goals, additional development has yieldeda new valve design, spring-loaded tensioning motor heads, embeddedstrain gauge, and a multi-channel pump.

In the event that the IOM is fabricated from a gas-impermeable material,the IOM can include a gas-exchange membrane, membrane oxygenator, orRPPM-controlled gas injection.

The valve designs, as illustrated in FIGS. 5 and 9-13, utilize acircular ball bearing cage with microfluidic interconnects underneath.The compression pressure from the ball bearings acts to occlude thesemicrofluidic channels acting as a valve. The principal novel componentof this design is the placement of microfluidic channels inside thecircular path described by the rotating balls in the ball cage. Multipleconfigurations of this design are examined, each with very low deadfluid volume.

As shown in FIG. 9, the rotary planar valve (RPV) in this embodiment hasan actuator having a circular ball-bearing cage 910 defining a pluralityof equally spaced-apart openings 912 thereon, and a plurality of balls915 accommodated in the plurality of equally spaced-apart openings 912such that at least one opening accommodates no ball bearing. Each twoadjacent openings 912 through the center of the circular ball-bearingcage define an angle θ=2π/K, K being the number of the plurality ofequally spaced-apart openings.

Additionally, the RPV also has a plurality of selectively controllablechannels, e.g., 921-923, positioned under the actuator in relation tothe plurality of equally spaced-apart openings such that at least oneselectively controllable channel is positioned under the at least oneno-ball opening, for example, channel 922, so that a fluid flow isallowed through the open channel 922, while the other selectivelycontrollable channels 921 and 923 are respectively positioned under theopenings having the ball bearings so that no fluid flows are allowedthrough the other selectively controllable channels. For such a design,when rotating the actuator by a desired angle of (k×θ), k being 1, 2, .. . K, the at least one no-ball opening is selectively placed over adesired one of the selectively controllable channels.

Further, the RPV has at least one always-open channel, for example, 924a and 924 b positioned under the actuator in offset from the pluralityof equally spaced-apart openings, such that the offset channels 924 aand 924 b are in fluid communication with the selected open channel 922under the no-ball opening, while the other channels 921 and 923 underthe openings having the ball bearings 915 are closed. Two always-openchannels improve flow and ensure continuous flow during switching, ifdesired.

As shown in FIG. 9, each of the at least one always-open channels andthe plurality of selectively controllable channels has an end connectedto an arc fluidic path or a circular fluidic path 930.

For such an arrangement shown in FIG. 9, the ball bearings 915 occludechannel outputs from the valve; by convention, the circular ball-bearingcage 910 is rotated in increments of 45 degrees, and the pump drivingthe fluid is turned off while the circular ball-bearing cage 910 isbeing rotated; all controllable channels are on multiples of 45 degrees;always-open channels are offset by 22.5 degrees, i.e., 22.5+N*45degrees, and any missing ball allows a controllable channel to be open.

FIG. 10 shows different embodiments of the RPV according to theinvention. Each of the at least one always-open channels and theplurality of selectively controllable channels has an end connected toan arc fluidic path, as shown in FIG. 10A, or a circular fluidic path,as shown in FIG. 10B.

As shown in FIG. 11, four selectively controllable channels 1121-1124are connected to corners of a square fluidic path 1130, as shown in FIG.11A, and the actuator 1110 is configured such that when rotating by thedesired angle of (k×θ), the top-left and top-right channels 1121 and1124 are in fluid communication with each other through the top portion1134 of the square fluidic path 1130, and the bottom-left andbottom-right channels 1122 and 1123 are in fluid communication with eachother through the bottom portion 1132 of the square fluidic path 1130,as shown in FIG. 11C; or the top-left and bottom-left channels 1121 and1122 are in fluid communication with each other through the left portion1131 of the square fluidic path 1130, and the top-right and bottom-rightchannels 1124 and 1123 are in fluid communication with each otherthrough the right portion 1133 of the square fluidic path 1130, as shownin FIG. 11B.

As shown in FIG. 12, three selectively controllable channels 1221-1223are connected in a T-like junction, and the actuator 1210 is configuredsuch that when rotating by a desired angle of (k×θ), two of the threechannels, for example, channels 1221 and 1223, are in fluidcommunication with each other, while the other channel, e.g., channel1222, is closed. This configuration allows fluid to enter from 1223 andexit through either 1221 or 1222. Alternatively, the top channel, 1223could be closed while fluid could flow freely between 1221 and 1222.

FIG. 13 shows the RPV according to another embodiment of the invention,which is corresponding to a double valve with a single actuator. Thedouble valve has first and second always-open channels 1324 and 1344positioned under the actuator 1310 in offset from the plurality ofequally spaced-apart openings 1312, a first plurality of selectivelycontrollable channels 1321, 1322, 1323 and 1325 and a second pluralityof selectively controllable channels 1341, 1342, 1343 and 1345. Thefirst always-open channel 1324 and the first plurality of selectivelycontrollable channels 1321, 1322, 1323 and 1325 are connected to a firstarc fluidic path 1331, and the second always-open channel 1344 and thesecond plurality of selectively controllable channels 1341, 1342, 1343and 1345 are connected to a second arc fluidic path 1332. The first andsecond arc fluidic paths 1331 and 1332 are arranged in a circle and notin fluid communication with each other. As such, in operation, the firstalways-open channel 1324 is selectively in fluid communication with oneof the first plurality of selectively controllable channels 1321, 1322,1323 and 1325, while the second always-open channel 1344 is selectivelyin fluid communication with one of the second plurality of selectivelycontrollable channels 1341, 1342, 1343 and 1345.

In one embodiment, the actuator utilized in the RPV can also be used inthe RPPM as a driving force of the pump. In addition, the RPPM also hasan input channel and an output channel positioned under the actuator inrelation to the plurality of equally spaced-apart openings such thatwhen the actuator is rotated, a fluid flow is pumped from the inputchannel to the output channel.

In one embodiment, as shown in FIG. 14, each of the RPVs and the RPPMsfurther comprises a motor for rotating the actuator incrementally by theangle θ. The motor comprises a spring-loaded tensioning motor head or aself-tensioning motor head to ensure that the proper compressive forceis delivered to the microfluidic channels. The self-tensioning motorhead comprises a cylinder body 1461, where the cylinder body 1461 hasone or more helically cut slits 1462 around an axis 1465 of the cylinderbody, or two or more horizontally cut slits 1464 alternatively in X andY directions, as shown in FIG. 14 to provide the requisite compressiveforce by the balls upon the PDMS, and to accommodate for differences ortime-variations in the thickness or stiffness of the PDMS.

In yet another embodiment shown in FIG. 15, a strain gauge 1590 isembedded within PDMS 1510. The ball beneath the strain gauge 1590induces strain, which is detectable with a Wheatstone bridge andappropriate amplification circuitry, and can be used to determine RPPMor RPV ball position. Accordingly, when the ball rolls over the gauge,the resistance changes and the ball position is inferred.

FIG. 16 shows the RPV and the RPPM as stand-alone units as related tothe embodiments of the invention.

As shown in FIG. 17, two channels 1720 and 1730 are employed in a PDMSRPPM pumphead 1710 for pumping different materials, e.g., air or vacuumin one channel and fluid in the other. The more channels and/or ballcircles there are, the more options there are. Channel widths can bemodulated to provide different pumping rates per revolution.

In addition, according to embodiments of the invention, other rotarymeans of actuation, for example, cam follower, wheels, and castervalves/pumps, etc., as shown in FIGS. 44-50, can also be utilized topractice the invention.

FIG. 44A shows schematically a design of one particular microfluidiccompatible rotary planar device with design features that can be usedeither for use as a pump or as a valve. Two key advantages of thisdesign are: 1) the critical pre-use tensioning of the roller ballsagainst the flexible membrane is easily achieved by simply placing aknown weight or force against the rigid pressure holding plate; and 2)the ball bearing cage is implemented as ball containing sockets directlyand rigidly attached to the drive pin. For pre-tensioning, onceappropriate pressure has been added (possibly via a calibrated donutshaped weight) then simply tightening the holding screws will establisha known compressive force underneath the ball bearings to actuate thedesired pump or valve functionality. The pressure transfer bearinglocated under the pressure holding plate acts to enable low frictionrotation of the Teflon or other low-friction drive bearing while at thesame time providing uniform downward force pressure on the Teflon drivebearing. Since the shaft rotation is rigidly linked to the Teflon drivebearing, it allows for direct transfer of the rotation delivered eitherfrom a motor or a hand crank via interface collar to the fluid drivingball bearings. When the central shaft is rotated, typically via a motoror a hand crank, the rotary force is transferred to a Teflon or otherlow friction material which holds individual ball bearings captive inball cages. Alternatively shafted roller bearings could be used totransfer force into the deformable membrane. A rotary encoder assemblycan be used to provide electronic verification of ball speed and preciseball location—a critical parameter when the device is utilized as arotary planar valve assembly.

FIG. 44B shows bottom views of a drive bearing indicating that (a) ballsand (b) rollers are housed within the sockets, according two embodimentsof the invention. The drive bearing is utilized in the rotary planardevice shown in FIG. 44A.

FIG. 45 shows a variation of the pumping module shown in FIGS. 44A and44B where pumping channels are fabricated in hard plastics and coveredwith a flexible membrane forming one of the microfluidic channel sides.The membrane allows for channel closure when pressure is delivered by arolling ball bearing. The hard plastic fluidic channel can be fashionedwith semi-circular cross section to facilitate valve sealing.

FIG. 46 shows various implementations of an axle-driven,cam-follower-bearing type actuator used to implement the RPPMs and RPVsaccording embodiments of the invention. The actuator has a cam 4610 anda plurality of cam followers 4620 spaced-equally mounted onto the cam4610, as shown in FIG. 46A. In another embodiment, the plurality of camfollowers 4620 mounted onto the cam 4610, but not spaced-equallymounted, as shown in FIG. 46B, where there is no cam follower mounted ata location 4630. Further, the location 4630 is installed with a positionindicator 4640, as shown in FIG. 46C.

FIG. 47 shows various implementations of an axle-driven, roller-bearingtype actuator used to implement the RPPMs and RPVs according embodimentsof the invention. The actuator has a wheel 4710 and a plurality ofrollers 4720 mounted into the spaced-equally sockets, as shown in FIG.47A. In another embodiment, one socket 4730 does not house a roller, asshown in FIG. 47B, where there is no cam follower mounted at a location4730. Alternatively, the socket 4730 is installed with a positionindicator 4740, as shown in FIG. 47C.

FIG. 48 shows the design of a roller or ball bearing caster pumpassembly that can be used to create a peristaltic pump when used inconjunction with a planar microfluidic channel covered by a flexiblemembrane. The design incorporates a roller bearing mounted at anapproximate 45 degree angle on a motor driven shaft. As the motor shaftrotates, the portion of the roller bearing in contact with the planarflexible membrane will trace a circular path on top of the embeddedfluidic channel. Only one rounded edge of the roller or ball bearingouter rim will be in compressional contact with the flexible membrane,and the rolling rim bearing action will exert minimal frictional slidingforce on the flexible membrane, thus creating as a very efficient longlived pump. This approximate 45 degree rotary caster design can also beused to provide rotary actuation of planar valve assemblies. Animportant feature of this design is that rotary shaft encoders can beeasily attached to the rigid shaft coupling to provide exact informationas to which portion of the circular arc contact region is currentlycompressed—thus facilitating exact control over planar fluid switchconnection modes. The device is identified as a “rotary caster actuator”for planar microfluidic pumps or planar microfluidic valves, which canbe used to compress flexible membranes for providing either pumping orvalving functionality. This device is comprised of a central shaft withan angularly mounted ball or roller bearing assembly that contains anouter rim which will roll along a circular path when the central shaftis rotated. This device provides low sliding frictional force and hencelow wear against the flexible membrane of a planar valve or pumpcomponent. Additionally, this device supports the use of standardizedrotary encoder assemblies for the purpose of determining exact valveactuation position information or exact rotary pump speed information.

In one embodiment, the pumping device with direct drive and encoderincludes (a) socket ball bearing cage made of low friction polymer, likea Teflon, directly attached to a rotary drive shaft; (b) pressureholding plate that is held in place by holding screws and transfers thetensioning pressure to the drive bearing via pressure transfer bearing;(c) pressure transfer bearing that can be either stand alone part or beintegral part of the drive bearing; (d) rotary encoder; and (e)interface collar to provide attachment of a drive motor or a hand crank.In operation, microfluidic channels are located within the flexiblemembrane and placed under the device. In one embodiment, the device canbe used as a manually or attached motor actuated rotary valve with theattached encoder proving feedback indication of the ball bearingposition.

In one embodiment, the microfluidic channels are fabricated in hardplastic and are sealed with a flexible membrane with the ball bearing ofthe drive bearing acting on the flexible membrane.

FIG. 49 shows the conceptual design of a spring loaded pressure inverterwhich is comprised of a rotary array of actuators that can be used toprovide a plurality of normally-closed fluidic channel connections whichare driven by a central motorized rotary device, such as thatillustrated in FIG. 48, or alternatively by any of the ball bearing cageRPV actuators described previously. The device operates on the basis ofan embedded or otherwise rigidly mounted fulcrum which can transform thedownward pressure associated with a tensioned ball bearing, or rollerbearing into an upward force that can open a normally closedmicrofluidic valve. In this conceptual visualization eight NormallyClosed (N.C.) microfluidic valves are located in the central region ofthe planar assembly. The device assembly identified as a “spring loadedpressure inverter” which can be utilized to convert the downwardpressure exerted by a ball or roller bearing rotary actuator assemblyinto an upward force capable of opening a normally closed microfluidicchannel.

FIG. 50 shows additional views of the conceptual design for a springloaded pressure inverter actuation device for opening normally closedmicrofluidic valves. Note that the illustrated conceptual compressionsprings provide a force which is translated via a fulcrum mounted leverto provide the downward force that keeps a microfluidic channel closed.Only when external force is applied to a lever by a rotary actuatorelement will the associated microfluidic valve location be opened. Noteespecially that this is a conceptual diagram, and that actual physicalimplementation of the assembly might utilize flexible elastomers toprovide spring forces and one-piece integrated fulcrum flexure units toact as levers.

The principal novel features of the IOM Chip devices and theirrespective carriers/cartridges, as illustrated in FIGS. 28-36, are:

-   (1) In various implementations of the Chip Carrier/Organ Cartridge,    multiple fluidic reservoirs and either multiple pumps, or pumps used    in conjunction with fluidic input and/or output valves, could be    used to deliver various combinations of cell media, drugs, and other    liquid compounds to the biological material in the chip.-   (2) In other embodiments of the Chip Carrier/Cartridge, multiple    fluidic reservoirs and either multiple pumps, or pumps used in    conjunction with multi-position fluidic input valves, could be used    in order to deliver various combinations of cell media, drugs, and    other liquid compounds to the biological material in the chip. These    reservoirs can be integral to the chip, with either a distensible    boundary to allow filling or emptying, or a fixed volume with a    bacteriostatic air filter to allow the introduction of air as fluid    is removed by the on-chip pump, and vice versa.-   (3) In other embodiments of the Chip Carrier/Cartridge, multiple    fluidic reservoirs and either multiple pumps, or pumps used in    conjunction with multi-position fluidic input valves and bubble    traps, could be used to deliver various combinations of cell media,    drugs, and other liquid compounds to the biological material in the    chip.-   (4) A key feature of the Invented Chip Carrier/Cartridge is the    inclusion of an electronic microcontroller and an on-board battery.    The purpose of the microcontroller is to provide signals to the    pump(s) and fluidic valve assemblies that feed the biological    material in the Organ Chip assembly, and utilize other sensors to    control processes and/or conditions on the Chip Carrier/Cartridge.    The purpose of the battery is to allow autonomous operation of the    pumps and valves in the carrier when it has been disconnected from    its standard power supply, for example, during transport or while    being examined microscopically or chemically.-   (5) A key feature of the Invented Chip Carrier/Organ Cartridge/IOM    Chip interface that comprises the Organ Cartridge is the pump and    valve fluidic driving heads that sit on the surface of the TOM Chip.    The mechanism of operation of these pumps and valves is to occlude    channels in the flexible surface layer of the IOM Chip and either    move fluid in the case of the pumps or occlude fluid flow in the    case of valves.-   (6) A key feature of the Invented Chip Carrier/Cartridge is the    ability of the microcontroller to implement complicated fluid and    drug delivery protocols to the biological material on the chip. The    protocols can be preloaded into the microcontroller and the chip    carrier/cartridge can implement these fluid delivery instructions    without human intervention.-   (7) The microcontroller can also make autonomous decisions regarding    pumps and valves based upon the output of embedded sensors.-   (8) A key feature of the Invented Chip Carrier/Cartridge design is    the closed fluid delivery system which can be sterilized without    requiring sterilization of the driving electronics, motors, and    sensors. This is extremely important in the context of long-term    cell culture experiments, as the sterile barrier is never breached    within the IOM Chip even while it is being transported, for example,    between incubator and microscope.-   (9) A key feature of the Invented Chip Carrier/Cartridge design is    the ability of this design to accommodate in-line and intra-device    fluid de-bubbler assemblies. These fluid de-bubbler assemblies are    designed to prevent entry of small bubbles into cell assemblies or    fluid delivery channels. These de-bubbling assemblies could be    stand-alone devices or integrated into a complete microfluidic    device.-   (10) A key feature of all implementations of the Invented TOM Chip    is that it has very low recirculating dead volume which allows for    faster accumulation of cell signaling factors and avoidance of their    dilution to levels below what is required for physiological effects.-   (11) A key feature of the basic implementations of the Invented IOM    Chip is that all fluidic connections between the pumps and valves    are fabricated internally. The only external connections are to the    stock solutions, to other organs in a coupled microphysiological    system, or to storage vials for waste or analysis.-   (12) A key advantage of the Invented Chip Carrier/Cartridge design    is that the fluidic system is completely enclosed. This offers the    additional advantage of allowing use in low humidity incubators,    which are less likely to corrode electrical contacts and less likely    to support fungal and bacterial contamination.-   (13) The Invented Chip Carrier/Cartridge could also include on-board    temperature regulation, so that the chips could be maintained at a    desired operating temperature, either above or below the ambient    temperature, without the need for an external incubator.-   (14) The Chip Carrier/Cartridge can include fluid flow and fluid    pressure sensors.

Bubbles are a common problem in microfluidic devices, and can beparticularly troubling if the device contains living cells. The rate ofbubble formation and growth depends in part upon whether the system isoperating under positive or negative pressure. Although it is possibleto run an entire system at a negative pressure relative to atmosphere,this would reduce the available gases in the media and could adverselyaffect the metabolism of the cells. Bubbles can form in negativepressure fluid channels because the dissolved gas will tend toaccumulate on any nucleation site within the channel. Positive pressureis more likely to prevent bubbles than negative as long as bubbles arenot already in the channel. Once a bubble is formed in a negativepressure system, it will grow continuously. In a positive pressuresystem, bubbles tend to reduce their size as the gas is pressed throughthe PDMS and their solubility is increased with pressure. It is possibleto reduce bubble formation during fluid loading and pumping by makingthe fluid channels hydrophilic. Temperature gradients also influencebubble formation within the microfluidic channels. It is feasible thatin a system with distributed sub-assemblies, temperature differencesbetween sub-assemblies can alter the solubility of gases in thesolutions and lead to spontaneous bubble formation.

To avoid the damage to cells from any bubbles that might be introducedinto a microfluidic Organ Chip by either the surfaces of the chip or bythe interfaces to perfusion controller or the microclinical analyzer, anin-line bubble trap is developed and utilized for long-term cellcultures in the perfusion controller and/or the microclinical analyzerin which bubbles are isolated from the primary flow by a forest ofposts. The bubbles rise to a closed chamber above the post forest and,if necessary, may be withdrawn through an external valve, either usingsystem pressure and a liquid-impermeable membrane or external suction,with the valve located either directly on the Organ Chip or integratedwith the perfusion controller. It is also possible to remove small airbubbles through PDMS by applying negative pressure.

FIGS. 18-22 show different embodiments of a bubble trap utilized in aperfusion controller and a microclinical analyzer of organ chips.

As shown in FIGS. 19A, 19B, 19C and 20A, the bubble trap has two levelsof microfluidic channels defining a fluidic compartment therebetweenwith a vertical via connecting the first and second microfluidicchannels; an optional bubble accumulation chamber can be placed abovethe via. This via; serves as a bubble withdrawal channel or a bubbleaccumulation chamber; and provided a hydrophobic gas exchange membraneis placed between the via and the bubble withdrawal channel, therebyseparating the fluidic compartment from the bubble. Shown in FIG. 19C isan alternative implementation of the straight channel bubble trap thatminimizes possible dead volume associated with the introduction ofbubble accumulation chamber within the flow path of the fluid within thevia.

As shown in FIG. 19D, the bubble trap comprises a microfluidic channelcontaining a dense forest of micro-pillars within the fluidic path whichact as bubble sieves to catch passing bubbles while providingalternative parallel paths for fluid to move freely beneath or aroundthem. Included in this design is a bubble accumulation chamber directlyover the micro-pillars designed to isolate the bubbles.

Further, as shown in FIGS. 18 and 19A, a ceiling of the bubbleaccumulation chamber is formed of a hydrophobic gas exchange membranethat allows for bubble removal either from passive diffusion into theatmosphere or from applied gentle vacuum.

As shown in FIG. 21, bubbles can be trapped using a two-layermicrofluidic architecture. The lower layer contains the fluidic input,with bubbles, that is distributed laterally by a flow splitter and theninto a wide forest of microposts that arrest the bubbles. The bubblesrise into the closed accumulation volume above the microposts, wherethey either diffuse into the PDMS or are drawn off occasionally througha separate port. The bubble-free fluid continues in the lower-layerchannels, past the posts, and then into the cell-culture region. Theadvantage of this approach, in contrast to the more common practice ofusing microfabricated filters or fences to block bubbles from downstreamcell culture regions, is that the low flow allows for better trapping ofbubbles because the bubbles accumulate directly above the posts and thusthe difficulty in clearing the bubbles from the filter/fence is avoided.With this approach, the accumulating bubbles do not impede fluid flowand can be drawn off whenever they fill the accumulation volume. It isimportant to realize that it is not desirable to load cells through thebubble trap, but they could be loaded either by reversing flow from theoutlet, or with a separate cell-loading port.

FIG. 22 shows another embodiment of the bubble trap, which iscorresponding to a somewhat more sophisticated design for a two-layerendothelial extravasation model, wherein cells roll, attach, and thenmigrate across a confluent layer of endothelial cells grown on amembrane with cell-permeable pores. (A) Block diagram of bubble trapcontaining bioreactor. The lower layer contains a splitter to evenlydistribute flow throughout the device and a forest of posts to arrestbubbles. Above the forest of posts is an accumulation volume into whichbubbles rise after being trapped. (B) Picture of fabricated cell culturebioreactor containing bubble trap. (C) A composite image of the bubbles(out of focus above the device) that have risen into the accumulationvolume above the posts, with flow passing undisturbed underneath asindicated by the in-focus time lapsetrails of fluorescent beadssuspended in fluid that is passing around the posts. (D) A test ofaccumulation of bubbles in the bubble trap by introducing a stream ofbubbles with a bubble generator coupled to the bubble trap. Practicalmaximum volume of bubbles is approximately 80% of the accumulationvolume. If the accumulation volume reaches capacity, a separate channel(not shown) can be used to aspirate them from the trap. This approachallows the trapping of bubbles in a two-compartment system with anintervening cell layer.

The de-bubbler assemblies (bubble traps) are designed to removemicroscopic bubbles from the fluid contained within the microfluidicdevices housed in IOM Chips. They can be either “stand alone” devicesconnected to the rest of the fluidic networks via tubing or directoverlay connections, or they can be integrated onto the microfluidicchips.

-   1) In its simplest implementation the bubble trap features two    microfluidic channels located at different levels and connected by a    vertical via and an accumulation chamber with a hydrophobic gas    exchange membrane placed above the via. The hydrophobic membrane    separates the fluidic compartment from the bubble withdrawal    channels. It allows the air to escape while maintaining a    fluid-impermeable hydrophobic barrier.-   2) In some instances the withdrawal of the bubble through the    membrane can be aided by applying a gentle vacuum on the air side.    Such a vacuum can be provided by an on-board RPPM.-   3) Another realization of the bubble trap consists of a microfluidic    channel containing a dense forest of micro-pillars (posts) within    the fluidic path that act as bubble sieves to catch passing bubbles    while providing alternative parallel paths for fluid to move freely    between them. Under certain conditions trapped bubbles will be    collected in the bubble accumulation chamber placed directly above    the pillars.-   4) In some instances the ceiling of the bubble accumulation area    includes a hydrophobic gas exchange membrane that would allow for    bubble removal either from passive diffusion into the atmosphere or    from actively applied gentle vacuum while preventing any liquid to    escape.-   5) In some instances (especially when handled liquid volumes must be    minimized) the hydrophobic gas exchange membrane with the vacuum    withdrawal network can be placed directly above the bubble-trapping    pillars forming a gas permeable/liquid impermeable ceiling.-   6) The hydrophobic properties of the trapping pillars and the gas    exchange membrane can be enhanced with a thin film coating.

In one aspect of the invention, a method for analyzing a plurality ofbio-objects includes the steps of providing a plurality of fluids;providing a fluidic network configured to be in fluid communication withthe plurality of bio-objects and the plurality of fluids, wherein thefluidic network comprises a plurality of fluidic switches, one or moreon-chip pumps and a plurality of fluidic paths connected therebetween;and controlling the plurality of fluidic switches and the one or moreon-chip pumps to selectively and individually perfuse at least one ofthe plurality of bio-objects with at least one of the plurality offluids at a predetermined perfusion flow rate and deliver an effluent ofthe at least one bio-object responsive to the perfusion to apredetermined outlet destination for analysis, recirculation, wasteexhaust, or input to other bio-objects of the plurality of bio-objects.

The fluidic network further comprises a microclinical analyzer fordetecting properties of the effluent of the at least one bio-object.

In one embodiment, the method further comprises the step of calibratingthe microclinical analyzer.

In another embodiment, the method also includes the step of measuring apressure drop across the at least one bio-object perfused with the atleast one fluid, so as to regulate the flow rate of the at least onefluid through the at least one bio-object at the predetermined perfusionrate.

Further, the method includes the step of removing bubbles generated inthe fluidic network.

Among other things, the invented Chip Carrier has, compared withFiberCell Systems Duet Pump hollow fiber bioreactor cartridges, at leastthe following advantages:

-   -   (a) The invented system allows more devices to reside in an        incubator by reducing the size of the controlling        electro-mechanical components.    -   (b) The invented system allows investigators to observe cells on        a microscope, which is crucial for long-term investigative        studies.    -   (c) The invented system provides convenient automatic drug        delivery.    -   (d) The invented system could readily provide oxygenation of        fluids at lower net volume.    -   (e) The invented system is compatible with many different        microfluidic bioreactor designs.    -   (f) The invented system has a smaller ratio of cell volume to        perfusate volume and can condition media more rapidly.    -   (g) The invented system is small enough to operate autonomously        on a microscope stage.    -   (h) The invented system provides convenient automatic drug        delivery with wireless control of the protocols.    -   (i) The on-board sensors of the invented system enable        closed-loop control of the bioreactor.    -   (j) The invented system can provide closed-loop control of        oxygen and CO₂ levels.    -   (k) A standardized footprint for sensors, pumps, and valves        could be utilized to allow the Organ Cartridge to support a wide        variety of Organ Chip configurations.    -   (l) The invented system utilizes fabricated, internal        interconnects to provide on-chip pathways for circulating fluid.    -   (m) The microfabricated channels are shorter and smaller when        compared against the FiberCell system.

In addition, the invented Chip Carrier has, compared with CellASIC ONIXbMicrofluidic Perfusion Platform, the advantages of:

-   -   (a) The invented system controller costs less than $150.00 per        Organ Cartridge.    -   (b) The invented system is compatible with many different        microfluidic bioreactor designs.    -   (c) The invented system is self-contained, except for the        long-term need for a source of electrical power to recharge the        on-carrier batteries and the need for fresh nutrients to replace        those metabolized by the bio-object.    -   (d) Multiple IOM Chip Carrier/Cartridges can operate        autonomously in either an incubator or a microscope.    -   (e) With the standardized footprint approach, the system can be        readily utilized with existing wellplate handling devices (i.e.        microscopes, incubators, etc.).    -   (f) Multiple inexpensive integrated controllers render single,        expensive, large controllers obsolete.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments were chosen and described in order to explain theprinciples of the invention and their practical application so as toactivate others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

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1. A perfusion controller usable for analysis associating with aplurality of bio-objects, each bio-object including an organ or a groupof cells, comprising: (a) a plurality of inlets for providing aplurality of fluids; (b) a plurality of outlets; and (c) a fluidicnetwork coupled between the plurality of inlets and the plurality ofoutlets and being in fluid communication with the plurality ofbio-objects, wherein the fluidic network comprises a plurality offluidic switches and one or more on-chip pumps adapted for selectivelyand individually perfusing at least one of the plurality of bio-objectswith at least one of the plurality of fluids at a predeterminedperfusion flow rate and delivering an effluent of the at least onebio-object responsive to the perfusion to a predetermined one of theplurality of outlets, wherein the plurality of outlets is coupled to atleast one of an analyzer, a waste port, one of the plurality ofbio-objects, and the fluidic switch network.
 2. The perfusion controllerof claim 1, further comprising a perfusion reservoir having a pluralityof containers for containing the plurality of fluids, respectively,wherein the plurality of containers is coupled to the plurality ofinlets for respectively providing the plurality of fluids.
 3. Theperfusion controller of claim 2, wherein the fluidic network furthercomprises a plurality of fluidic paths in fluid communication with theplurality of fluidic switches and the one or more on-chip pumps, andwherein each bio-object is disposed in a corresponding fluidic path. 4.The perfusion controller of claim 3, further comprising amicrocontroller for individually controlling the plurality of fluidicswitches and the one or more on-chip pumps of the fluidic network as soto control a flow rate of each fluidic path.
 5. The perfusion controllerof claim 4, wherein the microcontroller is provided with at least one ofa wireless communication protocol and a backup battery.
 6. The perfusioncontroller of claim 4, further comprising one or more sensors at leastcoupled to the at least one bio-object for measuring a pressure dropacross the at least one bio-object perfused with the at least one fluid,so as to regulate the flow rate of the at least one fluid through the atleast one bio-object at the predetermined perfusion rate.
 7. Theperfusion controller of claim 1, wherein each fluidic switch comprises avalve having at least one pole and a plurality of throws, wherein the atleast one pole is selectively operable in fluid communication with oneof the plurality of throws.
 8. The perfusion controller of claim 7,wherein the plurality of throws of the first fluidic switch of thefluidic switch network is respectively coupled to the plurality ofinlets for respectively receiving the plurality of fluids therefrom. 9.The perfusion controller of claim 8, wherein the plurality of throws ofthe last fluidic switch of the fluidic switch network is respectivelycoupled to the plurality of outlets for selectively delivering theeffluent of the at least one bio-object responsive to the perfusion tothe predetermined outlet.
 10. The perfusion controller of claim 7,wherein the fluidic network comprises first, second, and third fluidicswitches and an on-chip pump, wherein the first fluidic switch comprisesa one-pole four-throw valve, the second fluidic switch comprises atwo-pole three-throw valve, and the third fluidic switch comprises aone-pole four-throw valve.
 11. The perfusion controller of claim 10,wherein the plurality of bio-objects includes organ N−1, organ N, andorgan N+1, wherein the organ N−1 is coupled to the second fluidicswitch, the organ N is coupled between the second fluidic switch and theon-chip pump that in turn is coupled to the third fluidic switches, andthe organ N+1 is coupled to the second and third fluidic switches. 12.The perfusion controller of claim 1, wherein each fluidic switchcomprises a rotary planar valve (RPV) and each on-chip pump comprises arotary planar peristaltic micropump (RPPM), wherein each of the RPV andthe RPPM comprises an actuator.
 13. The perfusion controller of claim12, wherein the actuator comprises a circular ball-bearing cage defininga plurality of spaced-apart openings thereon, and a plurality of ballsaccommodated in the plurality of spaced-apart openings, wherein thenumber of the plurality of balls is same as that of plurality ofspaced-apart openings of the circular ball-bearing cage, such that eachopening of the circular ball-bearing cage accommodates a respectiveball, or the number of the plurality of balls is less than that ofplurality of spaced-apart openings of the circular ball-bearing cage,such that at least one opening accommodates no ball.
 14. The perfusioncontroller of claim 13, wherein the plurality of spaced-apart openingsis spaced-equally defined on the circular ball-bearing cage, whereineach two adjacent openings through the center of the circularball-bearing cage define an angle θ=2π/K, K being the number of theplurality of equally spaced-apart openings.
 15. The perfusion controllerof claim 14, wherein the RPV further comprises a plurality ofselectively controllable channels positioned under the actuator inrelation to the plurality of equally spaced-apart openings such that atleast one selectively controllable channel is positioned under the atleast one no-ball opening or under at least one no-ball location of thecircular ball-bearing cage, so that a fluid flow is allowed through theat least one selectively controllable channel, while the otherselectively controllable channels are respectively positioned under theopenings having the ball bearings so that no fluid flows are allowedthrough the other selectively controllable channels, wherein whenrotating the actuator by a desired angle of (k×θ), k being 1, 2, . . .K, the at least one no-ball opening or no-ball location is selectivelyplaced over a desired one of the selectively controllable channels. 16.The perfusion controller of claim 15, wherein the plurality ofselectively controllable channels comprises three selectivelycontrollable channels connected in a T-like junction, and wherein theactuator is configured such that when rotating by a desired angle of(k×θ), two of the three channels are in fluid communication with eachother, while the other channel is closed.
 17. The perfusion controllerof claim 15, wherein the plurality of selectively controllable channelscomprises four selectively controllable channels connected to corners ofa square fluidic path, and wherein the actuator is configured such thatwhen rotating by the desired angle of (k×θ), the first and secondchannels are in fluid communication with each other through the topportion of the square fluidic path, and the third and fourth channelsare in fluid communication with each other through the bottom portion ofthe square fluidic path; or the first and fourth channels are in fluidcommunication with each other through the left portion of the squarefluidic path, and the second and third channels are in fluidcommunication with each other through the right portion of the squarefluidic path.
 18. The perfusion controller of claim 15, wherein the RPVfurther comprises at least one always-open channel positioned under theactuator in offset from the plurality of equally spaced-apart openings,such that the at least one offset channel is in fluid communication withthe at least one selectively controllable channel under the at least oneno-ball opening or location, and the other selectively controllablechannels under the openings having the ball bearings are closed.
 19. Theperfusion controller of claim 18, wherein each of the at least onealways-open channel and the plurality of selectively controllablechannels has an end connected to an arc fluidic path or a circularfluidic path.
 20. The perfusion controller of claim 18, wherein the atleast one always-open channel has first and second always-open channelspositioned under the actuator in offset from the plurality of equallyspaced-apart openings, wherein the plurality of selectively controllablechannels comprises a first plurality of selectively controllablechannels and a second plurality of selectively controllable channels,wherein the first always-open channel and the first plurality ofselectively controllable channels are connected to a first arc fluidicpath, and the second always-open channel and the second plurality ofselectively controllable channels are connected to a second arc fluidicpath, wherein the first and second arc fluidic paths are arranged in acircle and not in fluid communication with each other, such that inoperation, the first always-open channel is selectively in fluidcommunication with one of the first plurality of selectivelycontrollable channels, while the second always-open channel isselectively in fluid communication with one of the second plurality ofselectively controllable channels.
 21. The perfusion controller of claim12, wherein the RPPM further comprises an input channel and an outputchannel positioned under the actuator in relation to the plurality ofequally spaced-apart openings such that when the actuator is rotated, afluid flow is pumped from the input channel to the output channel. 22.The perfusion controller of claim 12, wherein the actuator comprises awheel defining a plurality of spaced-apart sockets thereon in a circle,and a plurality of rollers accommodated in the plurality of spaced-apartsockets such that a rotation of the wheel causes the plurality ofrollers to rotate along the circle.
 23. The perfusion controller ofclaim 12, wherein the actuator comprises a cam, and a plurality ofcam-followers engaged with the cam such that a rotation of the camcauses the plurality of cam-followers to rotate along a circular path.24. The perfusion controller of claim 12, wherein each of the RPV andthe RPPM further comprises a motor for rotating the actuator.
 25. Theperfusion controller of claim 24, wherein the motor comprises aspring-loaded tensioning motor head or a self-tensioning motor head. 26.The perfusion controller of claim 25, wherein the self-tensioning motorhead comprises a cylinder body, wherein the cylinder body has one ormore helically cut slits around an axis of the cylinder body, or two ormore horizontally cut slits alternatively in X and Y directions.
 27. Theperfusion controller of claim 1, wherein each of the one or more on-chippumps comprises a pneumatically actuated peristaltic pump.
 28. Theperfusion controller of claim 1, wherein each of the one or more on-chippumps comprises a mechanically actuated peristaltic pump.
 29. Theperfusion controller of claim 3, wherein the fluidic network furthercomprises at least one bubble trap coupled to at least one fluidic pathfor removing bubbles therefrom.
 30. The perfusion controller of claim29, wherein the at least one bubble trap comprises: (a) first and secondmicrofluidic channels located at the same or different levels defining afluidic compartment therebetween; (b) a vertical via for connecting thefirst and second microfluidic channels or connecting both of thechannels to a bubble accumulation chamber; (c) a bubble accumulationarea placed above the vertical via; (d) a bubble withdrawal channelplaced over the vertical via directly or above the bubble accumulationchamber; and (e) a hydrophobic gas exchange membrane placed between thevia and the bubble withdrawal channel for separating the fluidiccompartment from the bubble withdrawal channels.
 31. The perfusioncontroller of claim 29, wherein the at least one bubble trap comprises(a) a microfluidic channel containing a dense forest of micro-pillarswithin the fluidic path that act as bubble sieves catching passingbubbles while providing alternative parallel paths for fluid to movefreely beneath or around them; (b) a bubble accumulation chamber formeddirectly over the micro-pillars; and (c) an optional bubble withdrawalchannel located above the bubble accumulation chamber, separated fromthe atmosphere by a gas exchange membrane.
 32. The perfusion controllerof claim 31, wherein a ceiling of the bubble accumulation chamber isformed of a hydrophobic gas exchange membrane that allows for bubbleremoval either due to passive diffusion into the atmosphere or due toactively applied gentle vacuum while preventing liquid to escape. 33.The perfusion controller of claim 1, wherein the desired fluids containdyes, a plurality of drugs, media or the like.
 34. The perfusioncontroller of claim 1, wherein the plurality of bio-objects is connectedto each other through the fluid bus in series, parallel, or acombination of them.
 35. The perfusion controller of claim 34, whereinthe at least one bio-object is operably bypassable from the other of theplurality of bio-objects.
 36. The perfusion controller of claim 1, beingformed integrally with an optically transparent material.
 37. A systemfor analysis of a plurality of bio-objects, each bio-object including anorgan or a group of cells, comprising a network of perfusion controllershaving a plurality of perfusion controllers as claimed in claim 1,wherein the plurality of perfusion controllers is arranged in an arrayfor perfusing the plurality of bio-objects individually orsimultaneously.
 38. The system of claim 37, wherein the plurality ofperfusion controllers is arranged in series, parallel, or a combinationof them.
 39. The system of claim 37, wherein combinations of theplurality of bio-objects and the plurality of perfusion controllers arethemselves arranged in series, parallel, or a combination of them toform the network of perfusion controllers.
 40. A method for analyzing aplurality of bio-objects, each bio-object including an organ or a groupof cells, comprising the steps of: (a) providing a plurality of fluids;(b) providing a fluidic network configured to be in fluid communicationwith the plurality of bio-objects and the plurality of fluids, whereinthe fluidic network comprises a plurality of fluidic switches, one ormore on-chip pumps and a plurality of fluidic paths connectedtherebetween; and (c) controlling the plurality of fluidic switches andthe one or more on-chip pumps to selectively and individually perfuse atleast one of the plurality of bio-objects with at least one of theplurality of fluids at a predetermined perfusion flow rate and deliveran effluent of the at least one bio-object responsive to the perfusionto a predetermined outlet destination for analysis, recirculation, wasteexhaust, or input to other bio-objects of the plurality of bio-objects.41. The method of claim 40, wherein the fluidic network furthercomprises a microclinical analyzer for detecting properties of theeffluent of the at least one bio-object.
 42. The method of claim 41,further comprising the step of calibrating the microclinical analyzer.43. The method of claim 40, further comprising the step of measuring apressure drop across the at least one bio-object perfused with the atleast one fluid, so as to regulate the flow rate of the at least onefluid through the at least one bio-object at the predetermined perfusionrate.
 44. The method of claim 40, further comprising the step ofremoving bubbles generated in the fluidic network. 45-112. (canceled)